Methods for treating conditions associated with c-fms

ABSTRACT

Antigen binding proteins that bind to human c-fms protein are provided. Nucleic acids encoding the antigen binding protein, vectors, and cells encoding the same are also provided. The antigen binding proteins can inhibit binding of c-fms to CSF-1, reduce monocyte migration into tumors, and reduce the accumulation of tumor-associated macrophages.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/195,169filed on Aug. 20, 2008, which claims priority to U.S. ProvisionalApplication No. 60/957,148 filed on Aug. 21, 2007, and U.S. ProvisionalApplication No. 61/084,588 filed on Jul. 29, 2008, the disclosures ofwhich are incorporated herein by reference in their entirety for allpurposes.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-1340-US-DIV_SequenceListing_ST25.txt, created May 8, 2012, which is324 KB in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

BACKGROUND

Many human and mouse tumor cell lines secrete the cytokine CSF-1 (ColonyStimulating Factor-1, also known as Macrophage-Colony StimulatingFactor, M-CSF) that in turn attracts, promotes the survival, andactivates monocyte/macrophage cells through the receptor c-fms (FelineMcDonough Strain). Tumor associated macrophages (TAMs) (also known astumor infiltrating macrophages (TIMs)) can be the major component of thetumor stroma comprising as much as 50% of the cell tumor mass. Kelly etal., 1988, Br. J. Cancer 57:174-177; Leek et al., 1994, J. Leukoc. Biol.56:423-435. In surveys of primary human tumors, there is widespreadevidence for CSF-1 mRNA expression. In addition, many studies havedemonstrated that elevated serum CSF-1, the number of TAMs, or thepresence of tissue CSF-1 and/or c-fms are associated with a poorprognosis for cancer patients.

TAMs support tumor growth, metastasis and survival by a variety ofmeans, including direct mitogenic activity on tumor cells throughsecretion of PDGF, TGF-β and EGF and metastasis through production ofECM-degrading enzymes (reviewed in Leek and Harris, 2002, J. MammaryGland Biol and Neoplasia 7:177-189 and Lewis and Pollard, 2006, CancerRes 66:605-612). Another important means of tumor support by TAMs is thecontribution to neo-vascularization of tumors via production of variousproangiogenic factors such as COX-2, VEGFs, FGFs, EGF, nitric oxide,angiopoietins, and MMPs. Dranoff et al., 2004, Nat. Rev. Cancer 4:11-22;MacMicking et al., 1997, Annu. Rev. Immunol. 15:323-350; Mantovani etal., 1992, Immunol. Today 13:265-270. In addition, CSF-1-derivedmacrophages can be immunosuppressive via production of various factorssuch as prostaglandins, indolamine 2,3 dioxigenase, nitric oxide, IL-10,and TGFβ. MacMicking et al., 1997, Annu. Rev. Immunol. 15:323-350;Bronte et al, 2001, J. Immunother. 24:431-446.

CSF-1 is expressed both as a membrane-bound and as a soluble cytokine(Cerretti et al., 1988, Mol. Immunol. 25:761-770; Dobbin et al., 2005,Bioinformatics 21:2430-2437; Wong et al., 1987, Biochem. Pharmacol.36:4325-4329) and regulates the survival, proliferation, chemotaxis andactivation of macrophages and their precursors (Bourette et al., 2000,Growth Factors 17:155-166; Cecchini et al., 1994, Development120:1357-1372; Hamilton, 1997, J. Leukoc. Biol. 62:145-155; Hume, 1985,Sci. Prog. 69:485-494; Sasmono and Hume, in: The innate immune responseto infection (eds. Kaufmann, S., Gordon, S. & Medzhitov, R.) 71-94 (ASMPress, New York, 2004); Ross and Auger, in: The macrophage (eds. Burke,B. & Lewis, C.) (Oxford University Press, Oxford, 2002)).

The cognate receptor, which is the c-fms proto-oncogene (also known asM-CSFR, CSF-1R or CD115), is a 165-kD glycoprotein with an associatedtyrosine kinase activity and belongs to the class III receptor tyrosinekinase family that includes PDGFR-α, PDGFR-β, VEGFR1, VEGFR2, VEGFR3,Flt3 and c-kit. Blume-Jensen and Hunter, 2001, Nature 411:355-365;Schlessinger and Ullrich, 1992, Neuron 9:383-391; Sherr et al., 1985Cell 41:665-676; van der Geer et al., 1994, Annu. Rev. Cell. Biol.10:251-337. The oncogenic form of c-fms, v-fms, which is carried by theMcDonough strain of feline sarcoma virus is mutated to conferconstitutively activated protein kinase activity (Sherr et al., 1985,Cell 41:665-676; Roussel and Sherr, 2003, Cell Cycle 2: 5-6). Expressionof c-fms in normal cells is restricted to myelomonocytic cells(including monocytes, tissue macrophages, Kupffer cells, Langerhanscells, microglial cells and osteoclasts), hematopoietic precursors andtrophoblasts. Arai et al., 1999, J. Exp. Med. 190:1741-1754; Dai et al.,2002, Blood 99:111-120; Pixley and Stanley, 2004, Trends Cell Biol.14:628-638. Expression of c-fms has also been demonstrated in some tumorcells (Kirma et al., 2007, Cancer Res 67:1918-1926). A variety of invitro studies and analyses of mutant mice demonstrate that CSF-1 is aligand for c-fms (see, e.g., Bourette and Rohrschneider, 2000, GrowthFactors 17:155-166; Wiktor-Jedrzejczak et al., 1990, Proc. Natl. Acad.Sci. U.S.A. 87:4828-4832; Yoshida et al., 1990, Nature 345:442-444; vanWesenbeeck and van Hul, 2005, Crit. Rev. Eukaryot. Gene Expr.15:133-162). Binding of CSF-1 to c-fms induces autophosphorylation ofthe receptor at particular sites that result in downstream activation ofsignaling pathways including PI3-K/AKT and Ras/Raf/MEK/MAPK andmacrophage differentiation is mediated primarily through persistent MEKactivity (Gosse et al., 2005, Cellular Signaling 17:1352-1362). Veryrecent evidence indicates that interleukin-34 (IL-34) is also a ligandfor c-fms (Lin, et al. 2008, Science 320:807-811).

SUMMARY

Antigen-binding proteins that bind c-fms, including human c-fms, aredescribed herein. The human c-fms antigen-binding proteins were found toinhibit, interfere with, or modulate at least one of the biologicalresponses related to c-fms, and, as such, are useful for amelioratingthe effects of c-fms-related diseases or disorders. Binding of certainantigen-binding proteins to c-fms can, therefore, have one or more ofthe following activities: inhibiting, interfering with, or modulatingc-fms-CSF-1 binding or signaling, inhibiting c-fms-IL-34 binding orsignaling, reducing monocyte migration into tumors, and/or reducing theaccumulation of tumor-associated macrophages (TAMs).

One embodiment includes expression systems, including cell lines, forthe production of c-fms receptor antigen binding proteins and methodsfor diagnosing and treating diseases related to human c-fms.

Some of the isolated antigen-binding proteins that are describedcomprise (A) one or more heavy chain complementary determining regions(CDRHs) selected from the group consisting of: (i) a CDRH1 selected fromthe group consisting of SEQ ID NOs:136-147; (ii) a CDRH2 selected fromthe group consisting of SEQ ID NOs:148-164; (iii) a CDRH3 selected fromthe group consisting of SEQ ID NOs:165-190; and (iv) a CDRH of (i), (ii)and (iii) that contains one or more amino acid substitutions, deletionsor insertions that collectively total no more than four amino acids; (B)one or more light chain complementary determining regions (CDRLs)selected from the group consisting of: (i) a CDRL1 selected from thegroup consisting of SEQ ID NOs:191-210; (ii) a CDRL2 selected from thegroup consisting of SEQ ID NOs:211-224; (iii) a CDRL3 selected from thegroup consisting of SEQ ID NOs:225-246; and (iv) a CDRL of (i), (ii) and(iii) that contains one or more amino acid substitutions, deletions orinsertions that collectively total no more than four amino acids; or (C)one or more heavy chain CDRHs of (A) and one or more light chain CDRLsof (B).

In one embodiment, the isolated antigen-binding protein may comprise atleast one or two CDRH of the above-mentioned (A) and at least one or twoCDRL of the above-mentioned (B). In yet another aspect, the isolatedantigen-binding protein includes a CDRH1, a CDRH2, a CDRH3, a CDRL1, aCDRL2 and a CDRL3.

In certain antigen binding proteins, the CDRH of the above-mentioned (A)is further selected from the group consisting of: (i) a CDRH1 selectedfrom the group consisting of SEQ ID NOs:136-147; (ii) a CDRH2 selectedfrom the group consisting of SEQ ID NOs:148-164; (iii) a CDRH3 selectedfrom the group consisting of SEQ ID NOs:165-190; and (iv) a CDRH of (i),(ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than two amino acids; the CDRL of theabove-mentioned (B) is selected from the group consisting of: (i) aCDRL1 selected from the group consisting of SEQ ID NOs:191-210; (ii) aCDRL2 selected from the group consisting of SEQ ID NOs:211-224; (iii) aCDRL3 amino acid sequence selected from the group consisting of SEQ IDNOs:225-246; and (iv) a CDRL of (i), (ii) and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more thantwo amino acids; or (C) one or more heavy chain CDRHs of (A) and one ormore light chain CDRLs of (B).

In yet another embodiment, the isolated antigen-binding protein maycomprise (A) a CDRH selected from the group consisting of (i) a CDRH1selected from the group consisting of SEQ ID NOs:136-147; (ii) a CDRH2selected from the group consisting of SEQ ID NOs:148-164; and (iii) aCDRH3 selected from the group consisting of SEQ ID NOs:165-190; (B) aCDRL selected from the group consisting of (i) a CDRL1 selected from thegroup consisting of SEQ ID NOs:191-210; (ii) a CDRL2 selected from thegroup consisting of SEQ ID NOs:211-224; and (iii) a CDRL3 selected fromthe group consisting of SEQ ID NOs:225-246; or (C) one or more heavychain CDRHs of (A) and one or more light chain CDRLs of (B). In oneembodiment, the isolated antigen-binding protein may include (A) a CDRH1of SEQ ID NOs:136-147, a CDRH2 of SEQ ID NOs:148-164, and a CDRH3 of SEQID NOs:165-190, and (B) a CDRL1 of SEQ ID NOs:191-210, a CDRL2 of SEQ IDNOs:211-224, and a CDRL3 of SEQ ID NOs:225-246. In another embodiment,the variable heavy chain (V_(H)) has at least 90% sequence identity withan amino acid sequence selected from the group consisting of SEQ IDNOs:70-101, and/or the variable light chain (V_(L)) has at least 90%sequence identity with an amino acid sequence selected from the groupconsisting of SEQ ID NOs:102-135. In a further embodiment, the V_(H) isselected from the group consisting of SEQ ID NOs:70-101, and/or theV_(L) is selected from the group consisting of SEQ ID NOs:102-135.

In another aspect, an isolated antigen binding protein is provided thatspecifically binds to an epitope containing the c-fms subdomains Ig-like1-1 and Ig-like 1-2 of human c-fms.

In yet another aspect, an isolated antigen binding protein is providedthat binds c-fms that comprises: (A) one or more heavy chain CDRs(CDRHs) selected from the group consisting of (i) a CDRH1 with at least80% sequence identity to SEQ ID NOs:136-147; (ii) a CDRH2 with at least80% sequence identity to SEQ ID NOs:148-164; and (iii) a CDRH3 with atleast 80% sequence identity to SEQ ID NOs:165-190; (B) one or more lightchain CDRs (CDRLs) selected from the group consisting of: (i) a CDRL1with at least 80% sequence identity to SEQ ID NOs:191-210; (ii) a CDRL2with at least 80% sequence identity to SEQ ID NOs:211-224; and (iii) aCDRL3 with at least 80% sequence identity to SEQ ID NOs:225-246; or (C)one or more heavy chain CDRHs of (A) and one or more light chain CDRLsof (B). In one embodiment, the isolated antigen-binding protein includes(A) one or more CDRHs selected from the group consisting of: (i) a CDRH1with at least 90% sequence identity to SEQ ID NOs:136-147; (ii) a CDRH2with at least 90% sequence identity to SEQ ID NOs:148-164; and (iii) aCDRH3 with at least 90% sequence identity to SEQ ID NOs:165-190; (B) oneor more CDRLs selected from the group consisting of: (i) a CDRL1 with atleast 90% sequence identity to SEQ ID NOs:191-210; (ii) a CDRL2 with atleast 90% sequence identity to SEQ ID NOs:211-224; and (iii) a CDRL3with at least 90% sequence identity to SEQ ID NOs:225-246; or (C) one ormore heavy chain CDRHs of (A) and one or more light chain CDRLs of (B).

Another embodiment is an isolated antigen-binding protein that bindsc-fms, the antigen-binding protein including one or a combination ofCDRs having the consensus sequences described below. Groups A, B, and Crefer to sequences derived from phylogenetically related clones. In oneaspect, the CDRs from the various groups may be mixed and matched. Inanother aspect, the antigen binding protein comprises two or more CDRHsfrom one and the same group A, B, or C. In again another aspect, theantigen binding protein comprises two or more CDRLs from the same groupA, B, or C. In again another aspect, the antigen binding proteincomprises at least two or three CDRHs, and/or at least two or threeCDRLs from the same group A, B, or C. The consensus sequences for thedifferent groups are as follows:

Group A:

(a) a CDRH1 of the generic formula GYTX₁TSYGIS (SEQ ID NO:307), whereinX₁ is selected from the group consisting of F and L; (b) a CDRH2 of thegeneric formula WISAYNGNX₁NYAQKX₂QG (SEQ ID NO:308), wherein X₁ isselected from the group consisting of T and P, and X₂ is selected fromthe group consisting of L and F; (c) a CDRH3 of the generic formulaX₁X₂X₃X₄X₄X₅FGEX₆X₇X₈X₉FDY (SEQ ID NO:309), wherein X₁ is selected fromthe group consisting of E and D, X₂ is selected from the groupconsisting of S and Q, X₃ is selected from the group consisting of G andno amino acid, X₄ is selected from the group consisting of L and noamino acid, X₅ is selected from the group consisting of W and G, X₆ isselected from the group consisting of V and L, X₇ is selected from thegroup consisting of E and no amino acid, X₈ is selected from the groupconsisting of G and no amino acid, and X₉ is selected from the groupconsisting of F and L; (d) a CDRL1 of the generic formulaKSSX₁GVLX₂SSX₃NKNX₄LA (SEQ ID NO:310), wherein X₁ is selected from thegroup consisting of Q and S, X₂ is selected from the group consisting ofD and Y, X₃ is selected from the group consisting of N and D, and X₄ isselected from the group consisting of F and Y; (e) a CDRL2 of thegeneric formula WASX₁RES (SEQ ID NO:311), wherein X₁ is selected fromthe group consisting of N and T; and f. a CDRL3 of the generic formulaQQYYX₁X₂PX₃T (SEQ ID NO:312), wherein X₁ is selected from the groupconsisting of S and T, X₂ is selected from the group consisting of D andT, and X₃ is selected from the group consisting of F and P.

Group B:

(a) a CDRH1 having the generic formula GFTX₁X₂X₃AWMS (SEQ ID NO:313),wherein X₁ is selected from the group consisting of F and V, X₂ isselected from the group consisting of S and N, and X₃ is selected fromthe group consisting of N and T; (b) a CDRH2 having the generic formulaRIKX₁KTDGX₂TX₃DX₄AAPVKG (SEQ ID NO:314), wherein X₁ is selected from thegroup consisting of S and T, X₂ is selected from the group consisting ofG and W, X₃ is selected from the group consisting of T and A, and X₄ isselected from the group consisting of Y and N; (c) a CDRH3 having thegeneric formula X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃YYGX₁₄DV (SEQ ID NO:315),wherein X₁ is selected from the group consisting of E, D and G, X₂ isselected from the group consisting of Y, L and no amino acid, X₃ isselected from the group consisting of Y, R, G and no amino acid, X₄ isselected from the group consisting of H, G, S and no amino acid, X₅ isselected from the group consisting of I, A, L and no amino acid, X₆ isselected from the group consisting of L, V, T, P and no amino acid, X₇is selected from the group consisting of T, V, Y, G, W and no aminoacid, X₈ is selected from the group consisting of G, V, S and T, X₉ isselected from the group consisting of S, T, D, N and G, X₁₀ is selectedfrom the group consisting of G, F, P, and Y, X₁₁ is selected from thegroup consisting of G, Y and N, X₁₂ is selected from the groupconsisting of V and Y, X₁₃ is selected from the group consisting of W, Sand Y, and X₁₄ is selected from the group consisting of M, T and V; (d)a CDRL1 having the generic formula QASQDIX₁NYLN (SEQ ID NO:316), whereinX₁ is selected from the group consisting of S and N; (e) a CDRL2 havingthe generic formula DX₁SNLEX₂ (SEQ ID NO:317), wherein X₁ is selectedfrom the group consisting of A and T, and X₂ is selected from the groupconsisting of T and P; and (f) a CDRL3 having the generic formulaQQYDX₁LX₂T (SEQ ID NO:318), wherein X₁ is selected from the groupconsisting of N and D, and X₂ is selected from the group consisting of Land I.

Group C:

(a) a CDRH1 having the generic formula GFTFX₁SYGMH (SEQ ID NO:319),wherein X₁ is selected from the group consisting of S and I; (b) a CDRH2having the generic formula VIWYDGSNX₁YYADSVKG (SEQ ID NO:320), whereinX₁ is selected from the group consisting of E and K; (c) a CDRH3 havingthe generic formula SSX₁X₂X₃YX₄MDV (SEQ ID NO:321), wherein X₁ isselected from the group consisting of G, S and W, X₂ is selected fromthe group consisting of N, D and S, X₃ is selected from the groupconsisting of Y and F, and X₄ is selected from the group consisting of Dand G; (d) a CDRL1 having the generic formula QASX₁DIX₂NX₃LN (SEQ IDNO:322), wherein X₁ is selected from the group consisting of Q and H, X₂is selected from the group consisting of S and N, and X₃ is selectedfrom the group consisting of F and Y; (e) a CDRL2 having the genericformula DASNLEX₁ (SEQ ID NO:323), wherein X₁ is selected from the groupconsisting of T and I; and (f) a CDRL3 having the generic formulaQX₁YDX₂X₃PX₄T (SEQ ID NO:324), wherein X₁ is selected from the groupconsisting of Q and R, X₂ is selected from the group consisting of N andD, X₃ is selected from the group consisting of L and F, and X₄ isselected from the group consisting of F, L and I.

In yet another embodiment, the isolated antigen binding proteindescribed hereinabove comprises a first amino acid sequence comprisingat least one CDRH and a second amino acid sequence comprising at leastone CDRL. In one embodiment, the first and the second amino acidsequences are covalently bonded to each other. In a further embodiment,the first amino acid sequence of the isolated antigen-binding proteinincludes the CDRH3 of SEQ ID NOs:165-190, CDRH2 of SEQ ID NOs:148-164,and CDRH1 of SEQ ID NOs:136-147, and the second amino acid sequence ofthe isolated antigen binding protein comprises the CDRL3 of SEQ IDNOs:225-246, CDRL2 of SEQ ID NOs:211-224, and CDRL1 of SEQ IDNOs:191-210.

In one aspect, the isolated antigen-binding proteins provided herein canbe a monoclonal antibody, a polyclonal antibody, a recombinant antibody,a human antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof. In anotherembodiment, the antibody fragment of the isolated antigen-bindingproteins can be an Fab fragment, an Fab′ fragment, an F(ab′)₂ fragment,an Fv fragment, a diabody, or a single chain antibody molecule. In afurther embodiment, the isolated antigen binding protein is a humanantibody and can be of the IgG1-, IgG2-, IgG3-, or IgG4-type.

In yet another aspect, the isolated antigen-binding protein can competefor binding to the extracellular portion of human c-fms with an antigenbinding protein of one of the isolated antigen-binding proteinsprovided. In one embodiment, the isolated antigen binding protein canreduce monocyte chemotaxis, inhibit monocyte migration into tumors,inhibit accumulation of tumor associated macrophage in a tumor orinhibit accumulation of macrophages in a disease tissue whenadministered to a patient.

In a further aspect, also provided are isolated nucleic acid moleculesthat encode the antigen-binding proteins that bind to c-fms. In someinstances, the isolated nucleic acid molecules are operably-linked to acontrol sequence.

In another aspect, also provided are expression vectors and host cellstransformed or transfected with the expression vectors that comprise theaforementioned isolated nucleic acid molecules that encodeantigen-binding proteins that can bind to c-fms.

In another aspect, also provided are methods of preparing theantigen-binding proteins that includes the step of preparing the antigenbinding protein from a host cell that secretes the antigen-bindingprotein.

In yet another aspect, a pharmaceutical composition is providedcomprising at least one of the aforementioned antigen-binding proteinsprovided and a pharmaceutically acceptable excipient. In one embodiment,the pharmaceutical composition may comprise an additional active agentthat is selected from the group consisting of a radioisotope,radionuclide, a toxin, or a therapeutic and a chemotherapeutic group.

Embodiments of the invention further provide a method for treating orpreventing a condition associated with c-fms in a patient, comprisingadministering to a patient an effective amount of at least one isolatedantigen-binding protein. In one embodiment, the condition is cancer thatis selected from the group consisting of breast cancer, prostate cancer,colorectal cancer, endometrial adenocarcinoma, leukemia, lymphoma,melanoma, esophageal squamous cell cancer, gastric cancer, astrocyticcancer, endometrial cancer, cervical cancer, bladder cancer, renalcancer, bladder cancer, lung cancer, and ovarian cancer.

In another aspect, the invention provides a method of inhibiting bindingof CSF-1 to the extracellular portion of c-fms in a patient comprisingadministering an effective amount of at least one antigen-bindingprotein provided herein.

In yet another aspect, also provided is a method of inhibitingautophosphorylation of human c-fms in a patient comprising administeringan effective amount of at least one antigen binding protein providedherein.

Further provided, as yet another aspect, is a method of reducingmonocyte chemotaxis in a patient comprising administering an effectiveamount of at least one antigen binding protein.

In one aspect, also provided is a method of inhibiting monocytemigration into tumors in a patient comprising administering an effectiveamount of at least one antigen binding protein.

In another aspect, also provided is a method of inhibiting accumulationof tumor associated macrophage in a tumor in a patient comprisingadministering an effective amount of at least one antigen bindingprotein.

These and other aspects will be described in greater detail herein. Eachof the aspects provided can encompass various embodiments providedherein. It is therefore anticipated that each of the embodimentsinvolving one element or combinations of elements can be included ineach aspect described. Other features, objects, and advantages of thedisclosed are apparent in the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a sequence comparison of the heavy chain variable regionsprovided herein. FIG. 1B shows a sequence comparison of the light chainvariable regions provided herein. The CDR and framework regions areindicated.

FIG. 2 shows the lineage analysis for 29 anti-c-fms hybridomas. Aminoacid sequences corresponding to either the variable heavy (V_(H)) orvariable light (V_(L)) domain of all cloned hybridomas were aligned andcompared to one another to resolve antibody diversity. Dendrogramsrepresenting these comparative alignments are shown wherein horizontalbranch length corresponds to the relative number of substitutions(differences) between any two sequences or sequence clades (groups ofclosely related sequences). Sequences grouped together for determinationof consensus sequences are indicated.

FIG. 3 demonstrates the inhibition of AML-5 proliferation by the varioushybridoma anti-c-fms supernatants. FIG. 3A shows AML-5 Bioassay withhybridoma anti-c-fms supernatans. FIG. 3B shows AML-5 bioassay withpurified recombinant anti-c-fms antibodies. AML-5 cells were incubatedwith 10 ng/ml CSF-1 in the presence of decreasing concentrations ofantibody. After 72 hours, cell proliferation was measured using AlamarBlue.

FIG. 4 shows a CynoBM assay with titration of c-fms antibodies in CSF-1.The inhibition of CSF-1-enriched cynomolgus bone marrow cellproliferation by the various hybridoma anti-c-fms supernatants isillustrated. Cynomolgus bone marrow cells were incubated with 10 ng/mlCSF-1 in the presence of decreasing concentrations of antibody. After 72hours, cell proliferation was measured using Alamar Blue.

FIG. 5 shows the inhibition of ligand-induced pTyr-c-fms by the IgG₂mAbs (PT, parent forms). 293T/c-fms cells were serum-starved for 1 hrand were treated with IgG₂ mAbs, 1.109, 1.2 or 2.360 (PT) and controlmAbs anti-c-fms 3-4A4 (non-blocking) and anti-h-CD39 M105 (non-specific)in titration series (1.0 to 0.0001 μg/ml) or at 1.0 μg/ml (controls).Cells were then stimulated with 50 ng/ml CSF-1 for 5 min at 37° C. Wholecell lysates were immunoprecipitated with anti-c-fms C20 as described.Western blots were probed with either anti-pTyr 4G10 (top panel) oranti-c-fms C20 (bottom panel) for detection of pTyr/c-fms and totalc-fms, respectively.

FIG. 6 compares the inhibition of ligand-induced pTyr-c-fms by IgG₂ mAbs(PT versus SM (somatic mutation cured) forms). 293T/c-fms cells wereserum-starved for 1 hr and were treated with IgG₂ mAbs, 1.109, 1.2 or2.360 (both PT or SM) and control mAbs anti-c-fms 3-4A4 (non-blocking)at 1.0 and 0.1 μg/ml. Cells were then stimulated with 50 ng/ml CSF-1 for5 min at 37° C. and whole cell lysates were immunoprecipitated withanti-c-fms C20 as described. Western blots were probed with eitheranti-pTyr 4G10 (top panel) or anti-c-fms C20 (bottom panel) fordetection of pTyr/c-fms and total c-fms, respectively.

FIG. 7 shows a western blot of an immunoprecipitation of c-fms by IgG₂mAbs (PT versus SM forms). Whole cell lysates of unstimulated 293T/c-fmscells were immunoprecipitated overnight at 4° C. using IgG₂ mAbs, 1.109,1.2 or 2.360 (both PT or SM forms) and anti-c-fms C20 at 2.5 μg/ml. Thewestern blot was probed with anti-c-fms C20 and anti-rabbit IgG/HRP.

FIG. 8 shows the amino sequence (SEQ ID NO:1) of the extracellulardomain region of human c-fms.

FIG. 9 shows western blots of immunoprecipitation of c-fms SNPs.Expression constructs of the indicated c-fms SNPs were constructed andtransiently expressed in 293T/c-fms cells. Unstimulated whole celllysates were then immunoprecipitated with each mAb and control Abs.Western blots were probed with c-fms H300 and anti-rabbit IgG/HRP.

FIG. 10 shows the diagram of human c-fms ECD (extracellular domain) andtruncated constructs. The avidin tag is fusioned in frame at the Nterminus of c-fms. The first and last four amino acids are indicated foreach c-fms constructs.

FIG. 11 demonstrates the binding of FITC labeled anti-avidin, 1.109, 1.2and 2.360 c-fms antibodies to c-fms ECD and truncated avidin fusionprotein.

FIG. 12 shows the binding of anti-avidin FITC, control antibody andanti-c-fms antibodies (FITC labeled) to full length c-fms and Ig-likeloop 2 (alone) fusion protein.

FIG. 13 exhibits the competition assay with 20× unlabeled 1.109, 1.2,and 2.360 c-fms antibodies, followed by 1 μg/ml concentration of FITClabeled 1.109.

FIG. 14 shows the competition assay with 20× unlabeled 1.109, 1.2, and2.360 c-fms antibodies, followed by 1 μg/ml concentration of FITClabeled 1.2.

FIG. 15 shows the competition assay with 20× unlabeled 1.109, 1.2, and2.360 c-fms antibodies, followed by 1 μg/ml concentration of FITClabeled 2.360.

FIG. 16 shows the inhibition of the growth of MDAMB231 breastadenocarcinoma xenograft by anti-murine c-fms antibody by way ofmeasuring tumor volume and the percent necrosis of each tumor. Thepercent necrosis of each tumor was then calculated from thesemeasurements and shown in FIG. 16

FIG. 17 shows the inhibition of the growth of established NCIH1975 lungadenocarcinoma xenografts. Tumor measurements and treatment days areshown, demonstrating that an anti-murine c-fms antibody can inhibit thegrowth of an established NCIH1975 lung adenocarcinoma xenograft.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present application are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001), Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), which are incorporated herein byreference. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The terminology used in connection with,and the laboratory procedures and techniques of, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well known and commonly used in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the disclosed, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean ±1%.

DEFINITIONS

The term “polynucleotide” or “nucleic acid” includes bothsingle-stranded and double-stranded nucleotide polymers. The nucleotidescomprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Said modifications include base modifications such as bromouridine andinosine derivatives, ribose modifications such as 2′,3′-dideoxyribose,and internucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

The term “oligonucleotide” means a polynucleotide comprising 200 orfewer nucleotides. In some embodiments, oligonucleotides are 10 to 60bases in length. In other embodiments, oligonucleotides are 12, 13, 14,15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotidesmay be single stranded or double stranded, e.g., for use in theconstruction of a mutant gene. Oligonucleotides may be sense orantisense oligonucleotides. An oligonucleotide can include a label,including a radiolabel, a fluorescent label, a hapten or an antigeniclabel, for detection assays. Oligonucleotides may be used, for example,as PCR primers, cloning primers or hybridization probes.

An “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA,cDNA, or synthetic origin or some combination thereof which is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, or is linked to apolynucleotide to which it is not linked in nature. For purposes of thisdisclosure, it should be understood that “a nucleic acid moleculecomprising” a particular nucleotide sequence does not encompass intactchromosomes. Isolated nucleic acid molecules “comprising” specifiednucleic acid sequences may include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty otherproteins or portions thereof, or may include operably linked regulatorysequences that control expression of the coding region of the recitednucleic acid sequences, and/or may include vector sequences.

Unless specified otherwise, the left-hand end of any single-strandedpolynucleotide sequence discussed herein is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA transcriptthat are 5′ to the 5′ end of the RNA transcript are referred to as“upstream sequences;” sequence regions on the DNA strand having the samesequence as the RNA transcript that are 3′ to the 3′ end of the RNAtranscript are referred to as “downstream sequences.”

The term “control sequence” refers to a polynucleotide sequence that canaffect the expression and processing of coding sequences to which it isligated. The nature of such control sequences may depend upon the hostorganism. In particular embodiments, control sequences for prokaryotesmay include a promoter, a ribosomal binding site, and a transcriptiontermination sequence. For example, control sequences for eukaryotes mayinclude promoters comprising one or a plurality of recognition sites fortranscription factors, transcription enhancer sequences, andtranscription termination sequence. “Control sequences” can includeleader sequences and/or fusion partner sequences.

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein codinginformation into a host cell.

The term “expression vector” or “expression construct” refers to avector that is suitable for transformation of a host cell and containsnucleic acid sequences that direct and/or control (in conjunction withthe host cell) expression of one or more heterologous coding regionsoperatively linked thereto. An expression construct may include, but isnot limited to, sequences that affect or control transcription,translation, and, if introns are present, affect RNA splicing of acoding region operably linked thereto.

As used herein, “operably linked” means that the components to which theterm is applied are in a relationship that allows them to carry outtheir inherent functions under suitable conditions. For example, acontrol sequence in a vector that is “operably linked” to a proteincoding sequence is ligated thereto so that expression of the proteincoding sequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a gene of interest. The term includes the progeny of theparent cell, whether or not the progeny is identical in morphology or ingenetic make-up to the original parent cell, so long as the gene ofinterest is present.

The term “transduction” means the transfer of genes from one bacteriumto another, usually by bacteriophage. “Transduction” also refers to theacquisition and transfer of eukaryotic cellular sequences by replicationdefective retroviruses.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, BasicMethods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The term “transformation” refers to a change in a cell's geneticcharacteristics, and a cell has been transformed when it has beenmodified to contain new DNA or RNA. For example, a cell is transformedwhere it is genetically modified from its native state by introducingnew genetic material via transfection, transduction, or othertechniques. Following transfection or transduction, the transforming DNAmay recombine with that of the cell by physically integrating into achromosome of the cell, or may be maintained transiently as an episomalelement without being replicated, or may replicate independently as aplasmid. A cell is considered to have been “stably transformed” when thetransforming DNA is replicated with the division of the cell.

The terms “polypeptide” or “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms also apply to aminoacid polymers in which one or more amino acid residues is an analog ormimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers. The terms can also encompassamino acid polymers that have been modified, e.g., by the addition ofcarbohydrate residues to form glycoproteins, or phosphorylated.Polypeptides and proteins can be produced by a naturally-occurring andnon-recombinant cell; or it is produced by a genetically-engineered orrecombinant cell, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.The terms “polypeptide” and “protein” specifically encompass c-fmsantigen-binding proteins, antibodies, or sequences that have deletionsfrom, additions to, and/or substitutions of one or more amino acids ofan antigen-binding protein. The term “polypeptide fragment” refers to apolypeptide that has an amino-terminal deletion, a carboxyl-terminaldeletion, and/or an internal deletion as compared with the full-lengthprotein. Such fragments may also contain modified amino acids ascompared with the full-length protein. In certain embodiments, fragmentsare about five to 500 amino acids long. For example, fragments may be atleast 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350,400, or 450 amino acids long. Useful polypeptide fragments includeimmunologically functional fragments of antibodies, including bindingdomains. In the case of a c-fms-binding antibody, useful fragmentsinclude but are not limited to a CDR region, a variable domain of aheavy or light chain, a portion of an antibody chain or just itsvariable region including two CDRs, and the like.

The term “isolated protein” referred means that a subject protein (1) isfree of at least some other proteins with which it would normally befound, (2) is essentially free of other proteins from the same source,e.g., from the same species, (3) is expressed by a cell from a differentspecies, (4) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis associated in nature, (5) is operably associated (by covalent ornoncovalent interaction) with a polypeptide with which it is notassociated in nature, or (6) does not occur in nature. Typically, an“isolated protein” constitutes at least about 5%, at least about 10%, atleast about 25%, or at least about 50% of a given sample. Genomic DNA,cDNA, mRNA or other RNA, of synthetic origin, or any combination thereofmay encode such an isolated protein. Preferably, the isolated protein issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use.

A “variant” of a polypeptide (e.g., an antigen binding protein, or anantibody) comprises an amino acid sequence wherein one or more aminoacid residues are inserted into, deleted from and/or substituted intothe amino acid sequence relative to another polypeptide sequence.Variants include fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antigenbinding protein, or an antibody) that has been chemically modified insome manner distinct from insertion, deletion, or substitution variants,e.g., via conjugation to another chemical moiety.

The term “naturally occurring” as used throughout the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature.

An “antigen binding protein” as used herein means a protein thatspecifically binds a specified target antigen, such as c-fms or humanc-fms.

An antigen binding protein is said to “specifically bind” its targetantigen when the dissociation constant (K_(D)) is ≦10⁻⁸ M. The antibodyspecifically binds antigen with “high affinity” when the K_(D) is≦5×10⁻⁹ M, and with “very high affinity” when the K_(D) is ≦5×10⁻¹⁰ M.In one embodiment, the antibody has a K_(D) of ≦10⁻⁹ M and an off-rateof about 1×10⁻⁴/sec. In one embodiment, the off-rate is about1×10⁻⁵/sec. In other embodiments, the antibodies will bind to c-fms, orhuman c-fms with a K_(D) of between about 10⁻⁸M and 10⁻¹⁰ M, and in yetanother embodiment it will bind with a K_(D)≦2×10⁻¹⁰.

“Antigen binding region” means a protein, or a portion of a protein,that specifically binds a specified antigen. For example, that portionof an antigen binding protein that contains the amino acid residues thatinteract with an antigen and confer on the antigen binding protein itsspecificity and affinity for the antigen is referred to as “antigenbinding region.” An antigen binding region typically includes one ormore “complementary binding regions” (“CDRs”). Certain antigen bindingregions also include one or more “framework” regions. A “CDR” is anamino acid sequence that contributes to antigen binding specificity andaffinity. “Framework” regions can aid in maintaining the properconformation of the CDRs to promote binding between the antigen bindingregion and an antigen.

In certain aspects, recombinant antigen binding proteins that bind c-fmsprotein, or human c-fms, are provided. In this context, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as described herein.Methods and techniques for the production of recombinant proteins arewell known in the art.

The term “antibody” refers to an intact immunoglobulin of any isotype,or a fragment thereof that can compete with the intact antibody forspecific binding to the target antigen, and includes, for instance,chimeric, humanized, fully human, and bispecific antibodies. An“antibody” as such is a species of an antigen binding protein. An intactantibody generally will comprise at least two full-length heavy chainsand two full-length light chains, but in some instances may includefewer chains such as antibodies naturally occurring in camelids whichmay comprise only heavy chains. Antibodies may be derived solely from asingle source, or may be “chimeric,” that is, different portions of theantibody may be derived from two different antibodies as describedfurther below. The antigen binding proteins, antibodies, or bindingfragments may be produced in hybridomas, by recombinant DNA techniques,or by enzymatic or chemical cleavage of intact antibodies. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and mutations thereof,examples of which are described below.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable regiondomain, V_(L), and a constant region domain, C_(L). The variable regiondomain of the light chain is at the amino-terminus of the polypeptide.Light chains include kappa chains and lambda chains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable regiondomain, V_(H), and three constant region domains, C_(H)1, C_(H)2, andC_(H)3. The V_(H) domain is at the amino-terminus of the polypeptide,and the C_(H) domains are at the carboxyl-terminus, with the C_(H)3being closest to the carboxy-terminus of the polypeptide. Heavy chainsmay be of any isotype, including IgG (including IgG1, IgG2, IgG3 andIgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

The term “immunologically functional fragment” (or simply “fragment”) ofan antibody or immunoglobulin chain (heavy or light chain), as usedherein, is an antigen binding protein comprising a portion (regardlessof how that portion is obtained or synthesized) of an antibody thatlacks at least some of the amino acids present in a full-length chainbut which is capable of specifically binding to an antigen. Suchfragments are biologically active in that they bind specifically to thetarget antigen and can compete with other antigen binding proteins,including intact antibodies, for specific binding to a given epitope. Inone aspect, such a fragment will retain at least one CDR present in thefull-length light or heavy chain, and in some embodiments will comprisea single heavy chain and/or light chain or portion thereof. Thesebiologically active fragments may be produced by recombinant DNAtechniques, or may be produced by enzymatic or chemical cleavage ofantigen binding proteins, including intact antibodies. Immunologicallyfunctional immunoglobulin fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, Fv, domain antibodies and single-chain antibodies,and may be derived from any mammalian source, including but not limitedto human, mouse, rat, camelid or rabbit. It is contemplated further thata functional portion of the antigen binding proteins disclosed herein,for example, one or more CDRs, could be covalently bound to a secondprotein or to a small molecule to create a therapeutic agent directed toa particular target in the body, possessing bifunctional therapeuticproperties, or having a prolonged serum half-life.

An “Fab fragment” is comprised of one light chain and the C_(H)1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments comprising the C_(H)1and C_(H)2 domains of an antibody. The two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains.

An “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

An “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and5,260,203, the disclosures of which are incorporated by reference.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antigen binding protein” or “bivalent antibody” comprisestwo antigen binding sites. In some instances, the two binding sites havethe same antigen specificities. Bivalent antigen binding proteins andbivalent antibodies may be bispecific, see, infra.

A multispecific antigen binding protein” or “multispecific antibody” isone that targets more than one antigen or epitope.

A “bispecific,” “dual-specific” or “bifunctional” antigen bindingprotein or antibody is a hybrid antigen binding protein or antibody,respectively, having two different antigen binding sites. Bispecificantigen binding proteins and antibodies are a species of multispecificantigen binding protein or multispecific antibody and may be produced bya variety of methods including, but not limited to, fusion of hybridomasor linking of Fab′ fragments. See, e.g., Songsivilai and Lachmann, 1990,Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol.148:1547-1553. The two binding sites of a bispecific antigen bindingprotein or antibody will bind to two different epitopes, which mayreside on the same or different protein targets.

The term “neutralizing antigen binding protein” or “neutralizingantibody” refers to an antigen binding protein or antibody,respectively, that binds to a ligand, prevents binding of the ligand toits binding partner and interrupts the biological response thatotherwise would result from the ligand binding to its binding partner.In assessing the binding and specificity of an antigen binding protein,e.g., an antibody or immunologically functional fragment thereof, anantibody or fragment will substantially inhibit binding of a ligand toits binding partner when an excess of antibody reduces the quantity ofbinding partner bound to the ligand by at least about 20%, 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (as measured in anin vitro competitive binding assay). In the case of a c-fms antigenbinding proteins, such a neutralizing molecule will diminish the abilityof c-fms to bind CSF-1. In some embodiments, the neutralizing antigenbinding protein inhibits the ability of c-fms to bind IL-34. In otherembodiments, the neutralizing antigen binding protein inhibits theability of c-fms to bind CSF-1 and IL-34.

The term “compete” when used in the context of antigen binding proteins(e.g., neutralizing antigen binding proteins or neutralizing antibodies)that compete for the same epitope means competition between antigenbinding proteins is determined by an assay in which the antigen bindingprotein (e.g., antibody or immunologically functional fragment thereof)under test prevents or inhibits specific binding of a reference antigenbinding protein (e.g., a ligand, or a reference antibody) to a commonantigen (e.g., c-fms or a fragment thereof). Numerous types ofcompetitive binding assays can be used, for example: solid phase director indirect radioimmunoassay (RIA), solid phase direct or indirectenzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahliet al., 1983, Methods in Enzymology 9:242-253); solid phase directbiotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.137:3614-3619) solid phase direct labeled assay, solid phase directlabeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, ALaboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, etal., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer etal., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assayinvolves the use of purified antigen bound to a solid surface or cellsbearing either of these, an unlabelled test antigen binding protein anda labeled reference antigen binding protein. Competitive inhibition ismeasured by determining the amount of label bound to the solid surfaceor cells in the presence of the test antigen binding protein. Usuallythe test antigen binding protein is present in excess. Antigen bindingproteins identified by competition assay (competing antigen bindingproteins) include antigen binding proteins binding to the same epitopeas the reference antigen binding proteins and antigen binding proteinsbinding to an adjacent epitope sufficiently proximal to the epitopebound by the reference antigen binding protein for steric hindrance tooccur. Additional details regarding methods for determining competitivebinding are provided in the examples herein. Usually, when a competingantigen binding protein is present in excess, it will inhibit specificbinding of a reference antigen binding protein to a common antigen by atleast 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance,binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as an antigenbinding protein (including, e.g., an antibody or immunologicalfunctional fragment thereof), and additionally capable of being used inan animal to produce antibodies capable of binding to that antigen. Anantigen may possess one or more epitopes that are capable of interactingwith different antigen binding proteins, e.g., antibodies.

The term “epitope” is the portion of a molecule that is bound by anantigen binding protein (for example, an antibody). The term includesany determinant capable of specifically binding to an antigen bindingprotein, such as an antibody or to a T-cell receptor. An epitope can becontiguous or non-contiguous (e.g., in a polypeptide, amino acidresidues that are not contiguous to one another in the polypeptidesequence but that within in context of the molecule are bound by theantigen binding protein). In certain embodiments, epitopes may bemimetic in that they comprise a three dimensional structure that issimilar to an epitope used to generate the antigen binding protein, yetcomprise none or only some of the amino acid residues found in thatepitope used to generate the antigen binding protein. Most often,epitopes reside on proteins, but in some instances may reside on otherkinds of molecules, such as nucleic acids. Epitope determinants mayinclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl or sulfonyl groups, and may havespecific three dimensional structural characteristics, and/or specificcharge characteristics. Generally, antibodies specific for a particulartarget antigen will preferentially recognize an epitope on the targetantigen in a complex mixture of proteins and/or macromolecules.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) must be addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

In calculating percent identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Thecomputer program used to determine percent identity is the GCG programpackage, which includes GAP (Devereux et al., 1984, Nucl. Acid Res.12:387; Genetics Computer Group, University of Wisconsin, Madison,Wis.). The computer algorithm GAP is used to align the two polypeptidesor polynucleotides for which the percent sequence identity is to bedetermined. The sequences are aligned for optimal matching of theirrespective amino acid or nucleotide (the “matched span”, as determinedby the algorithm). A gap opening penalty (which is calculated as 3× theaverage diagonal, wherein the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. In certain embodiments, a standard comparison matrix (see,Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl.Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) isalso used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program are the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4

Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (GAP program) canbe adjusted if so desired to result in an alignment that spans at least50 contiguous amino acids of the target polypeptide.

As used herein, “substantially pure” means that the described species ofmolecule is the predominant species present, that is, on a molar basisit is more abundant than any other individual species in the samemixture. In certain embodiments, a substantially pure molecule is acomposition wherein the object species comprises at least 50% (on amolar basis) of all macromolecular species present. In otherembodiments, a substantially pure composition will comprise at least80%, 85%, 90%, 95%, or 99% of all macromolecular species present in thecomposition. In other embodiments, the object species is purified toessential homogeneity wherein contaminating species cannot be detectedin the composition by conventional detection methods and thus thecomposition consists of a single detectable macromolecular species.

The term “treating” refers to any indicia of success in the treatment oramelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the injury, pathology or conditionmore tolerable to the patient; slowing in the rate of degeneration ordecline; making the final point of degeneration less debilitating;improving a patient's physical or mental well-being. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. For example,certain methods presented herein successfully treat cancer by decreasingthe incidence of cancer, causing remission of cancer and/or amelioratinga symptom associated with cancer or an inflammatory disease.

An “effective amount” is generally an amount sufficient to reduce theseverity and/or frequency of symptoms, eliminate the symptoms and/orunderlying cause, prevent the occurrence of symptoms and/or theirunderlying cause, and/or improve or remediate the damage that resultsfrom or is associated with cancer. In some embodiments, the effectiveamount is a therapeutically effective amount or a prophylacticallyeffective amount. A “therapeutically effective amount” is an amountsufficient to remedy a disease state (e.g. cancer) or symptoms,particularly a state or symptoms associated with the disease state, orotherwise prevent, hinder, retard or reverse the progression of thedisease state or any other undesirable symptom associated with thedisease in any way whatsoever. A “prophylactically effective amount” isan amount of a pharmaceutical composition that, when administered to asubject, will have the intended prophylactic effect, e.g., preventing ordelaying the onset (or reoccurrence) of cancer, or reducing thelikelihood of the onset (or reoccurrence) of cancer or cancer symptoms.The full therapeutic or prophylactic effect does not necessarily occurby administration of one dose, and may occur only after administrationof a series of doses. Thus, a therapeutically or prophylacticallyeffective amount may be administered in one or more administrations.

“Amino acid” includes its normal meaning in the art. The twentynaturally-occurring amino acids and their abbreviations followconventional usage. See, Immunology—A Synthesis, 2nd Edition, (E. S.Golub and D. R. Green, eds.), Sinauer Associates: Sunderland, Mass.(1991), incorporated herein by reference for any purpose. Stereoisomers(e.g., D-amino acids) of the twenty conventional amino acids, unnaturalamino acids such as [alpha]-, [alpha]-disubstituted amino acids, N-alkylamino acids, and other unconventional amino acids may also be suitablecomponents for polypeptides and are included in the phrase “amino acid.”Examples of unconventional amino acids include: 4-hydroxyproline,[gamma]-carboxyglutamate, [epsilon]-N,N,N-trimethyllysine,[epsilon]-N-acetyllysine, O-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,[sigma]-N-methylarginine, and other similar amino acids and imino acids(e.g., 4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxyl-terminal direction, in accordance withstandard usage and convention.

General Overview

Antigen-binding proteins that bind c-fms protein, including human c-fms(hc-fms) protein are provided herein. The antigen binding proteinsprovided are polypeptides into which one or more complementarydetermining regions (CDRs), as described herein, are embedded and/orjoined. In some antigen binding proteins, the CDRs are embedded into a“framework” region, which orients the CDR(s) such that the properantigen binding properties of the CDR(s) is achieved. In general,antigen binding proteins that are provided can interfere with, block,reduce or modulate the interaction between CSF-1 and c-fms.

Certain antigen binding proteins described herein are antibodies or arederived from antibodies. In certain embodiments, the polypeptidestructure of the antigen binding proteins is based on antibodies,including, but not limited to, monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), and fragmentsthereof. The various structures are further described herein below.

The antigen binding proteins provided herein have been demonstrated tobind to the extracellular domain of c-fms, in particular human c-fms. Asdescribed further in the examples below, certain antigen bindingproteins were tested and found to bind to epitopes different from thosebound by a number of other anti-c-fms antibodies. The antigen bindingproteins that are provided compete with CSF-1 and thereby prevent CSF-1from binding to its receptor. In certain embodiments, antigen bindingproteins inhibit binding between IL-34 and c-fms. In other embodiments,the antigen binding proteins inhibit the ability of c-fms to bind bothCSF-1 and IL-34. As a consequence, the antigen binding proteins providedherein are capable of inhibiting c-fms activity. In particular, antigenbinding proteins binding to these epitopes can have one or more of thefollowing activities: inhibiting, inter alia, c-fms autophosphorylation,induction of c-fms signal transduction pathways, c-fms induced cellgrowth, monocyte chemotaxis accumulation of tumor associated macrophagesin a tumor or in the stroma of a tumor, production of tumor-promotingfactors and other physiological effects induced by c-fms upon CSF-1binding. The antigen binding proteins that are disclosed herein have avariety of utilities. Some of the antigen binding proteins, forinstance, are useful in specific binding assays, affinity purificationof c-fms, in particular hc-fms or its ligands and in screening assays toidentify other antagonists of c-fms activity. Some of theantigen-binding proteins are useful for inhibiting binding of CSF-1 toc-fms, or inhibiting autophosphorylation of c-fms.

The antigen-binding proteins can be used in a variety of treatmentapplications, as explained herein. For example, certain c-fmsantigen-binding proteins are useful for treating conditions associatedwith c-fms, such as reducing monocyte chemotaxis in a patient,inhibiting monocyte migration into tumors, inhibiting accumulation oftumor associated macrophage in a tumor or inhibiting angiogenesis, as isfurther described herein. In certain embodiments, the antigen bindingproteins inhibit the ability of TAMs to promote tumor growth,progression and/or metastasis. In addition, in cases where the tumorcells themselves express and use c-fms, antibody binding to c-fms couldinhibit their growth/survival. Other uses for the antigen bindingproteins include, for example, diagnosis of c-fms-associated diseases orconditions and screening assays to determine the presence or absence ofc-fms. Some of the antigen binding proteins described herein are usefulin treating consequences, symptoms, and/or the pathology associated withc-fms activity. These include, but are not limited to, various types ofcancer and inflammatory disease and well as cancer cachexia. In someembodiments, the antigen binding proteins can be used to treat variousbone disorders.

C-fms

Colony-stimulating factor 1 (CSF-1) promotes the survival,proliferation, and differentiation of mononuclear phagocyte lineages.CSF-1 exerts its activities by binding to the cell-surface c-fmsreceptor, resulting in autophosphorylation by receptor c-fms kinase anda subsequent cascade of intracellular signals.

The terms “c-fms,” “c-fms receptor,” “human c-fms”, and “human c-fmsreceptor” refer to a cell surface receptor that binds to a ligand,including, but not limited to, CSF-1 and as a result initiates a signaltransduction pathway within the cell. In some embodiments, the receptorcan bind IL-34 or both CSF-1 and IL-34. The antigen binding proteinsdisclosed herein bind to c-fms, in particular human c-fms. An exemplaryextracellular domain of human c-fms amino acid sequence is depicted inSEQ ID NO:1. As described below, c-fms proteins may also includefragments. As used herein, the terms are used interchangeably to mean areceptor, in particular a human receptor that binds specifically toCSF-1.

The term human c-fms (h-cfms) receptor as used herein also includesnaturally occurring alleles, including the mutations A245S, V279M andH362R. The term c-fms also includes post-translational modifications ofthe c-fms amino acid sequence. For example, the extracellular domain(ECD) of human c-fms (residues 20-512 of the receptor) has elevenpossible N-linked glycosylation sites in the sequence. Thus, the antigenbinding proteins may bind to or be generated from proteins glycosylatedat one or more of the positions.

The c-fms signal transduction pathway is up-regulated in a number ofhuman pathologies that involve chronic activation of tissue macrophagepopulations. Increases in CSF-1 production are also associated with theaccumulation of tissue macrophages seen in various inflammatory diseasessuch as inflammatory bowel disease. In addition, the growth of severaltumor types is associated with overexpression of CSF-1 and c-fmsreceptor in cancer cells and/or tumor stroma.

C-fms Receptor Antigen Binding Proteins

A variety of selective binding agents useful for regulating the activityof c-fms are provided. These agents include, for instance, antigenbinding proteins that contain an antigen binding domain (e.g., singlechain antibodies, domain antibodies, immunoadhesions, and polypeptideswith an antigen binding region) and specifically bind to a c-fmspolypeptide, in particular human c-fms. Some of the agents, for example,are useful in inhibiting the binding of CSF-1 to c-fms, and can thus beused to inhibit, interfere with or modulate one or more activitiesassociated with c-fms signaling. In certain embodiments, the antigenbinding proteins can be used to inhibit binding between IL-34 and c-fms.In some embodiments, the antigen binding proteins interfere with theability of c-fms to bind both CSF-1 and IL-34.

In general, the antigen binding proteins that are provided typicallycomprise one or more CDRs as described herein (e.g., 1, 2, 3, 4, 5 or6). In some instances, the antigen binding protein comprises (a) apolypeptide structure and (b) one or more CDRs that are inserted intoand/or joined to the polypeptide structure. The polypeptide structurecan take a variety of different forms. For example, it can be, orcomprise, the framework of a naturally occurring antibody, or fragmentor variant thereof, or may be completely synthetic in nature. Examplesof various polypeptide structures are further described below.

In certain embodiments, the polypeptide structure of the antigen bindingproteins is an antibody or is derived from an antibody, including, butnot limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto herein as “antibody mimetics”), chimeric antibodies, humanizedantibodies, antibody fusions (sometimes referred to as “antibodyconjugates”), and portions or fragments of each, respectively. In someinstances, the antigen binding protein is an immunological fragment ofan antibody (e.g., a Fab, a Fab′, a F(ab′)₂, or a scFv). The variousstructures are further described and defined herein.

Certain of the antigen binding proteins as provided herein specificallybind to human c-fms. In a specific embodiment, the antigen bindingprotein specifically binds to human c-fms protein having the amino acidsequence of SEQ ID NO:1.

In embodiments where the antigen binding protein is used for therapeuticapplications, an antigen binding protein can inhibit, interfere with ormodulate one or more biological activities of c-fms. In this case, anantigen binding protein binds specifically and/or substantially inhibitsbinding of human c-fms to CSF-1 when an excess of antibody reduces thequantity of human c-fms bound to CSF-1, or vice versa, by at least about20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (forexample by measuring binding in an in vitro competitive binding assay).C-fins has many distinct biological effects, which can be measured inmany different assays in different cell types; examples of such assaysare provided herein.

Naturally Occurring Antibody Structure

Some of the antigen binding proteins that are provided have thestructure typically associated with naturally occurring antibodies. Thestructural units of these antibodies typically comprise one or moretetramers, each composed of two identical couplets of polypeptidechains, though some species of mammals also produce antibodies havingonly a single heavy chain. In a typical antibody, each pair or coupletincludes one full-length “light” chain (in certain embodiments, about 25kDa) and one full-length “heavy” chain (in certain embodiments, about50-70 kDa). Each individual immunoglobulin chain is composed of several“immunoglobulin domains”, each consisting of roughly 90 to 110 aminoacids and expressing a characteristic folding pattern. These domains arethe basic units of which antibody polypeptides are composed. Theamino-terminal portion of each chain typically includes a variabledomain that is responsible for antigen recognition. The carboxy-terminalportion is more conserved evolutionarily than the other end of the chainand is referred to as the “constant region” or “C region”. Human lightchains generally are classified as kappa and lambda light chains, andeach of these contains one variable domain and one constant domain.Heavy chains are typically classified as mu, delta, gamma, alpha, orepsilon chains, and these define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. IgG has several subtypes, including,but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes includeIgM, and IgM2. IgA subtypes include IgA1 and IgA2. In humans, the IgAand IgD isotypes contain four heavy chains and four light chains; theIgG and IgE isotypes contain two heavy chains and two light chains; andthe IgM isotype contains five heavy chains and five light chains. Theheavy chain C region typically comprises one or more domains that may beresponsible for effector function. The number of heavy chain constantregion domains will depend on the isotype. IgG heavy chains, forexample, each contain three C region domains known as C_(H)1, C_(H)2 andC_(H)3. The antibodies that are provided can have any of these isotypesand subtypes. In certain embodiments, the c-fms antibody is of the IgG1,IgG2, or IgG4 subtype.

In full-length light and heavy chains, the variable and constant regionsare joined by a “J” region of about twelve or more amino acids, with theheavy chain also including a “D” region of about ten more amino acids.See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989,New York: Raven Press (hereby incorporated by reference in its entiretyfor all purposes). The variable regions of each light/heavy chain pairtypically form the antigen binding site.

One example of an IgG2 heavy constant domain of an exemplary c-fmsmonoclonal antibody has the amino acid sequence:

(SEQ. ID NO: 2            ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*; asterisk corresponds to stop codon).

One example of a kappa light Constant domain of an exemplary c-fmsmonoclonal antibody has the amino acid sequence:

(SEQ ID NO: 3            RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*;asterisk corresponds to stop codon).

Variable regions of immunoglobulin chains generally exhibit the sameoverall structure, comprising relatively conserved framework regions(FR) joined by three hypervariable regions, more often called“complementarity determining regions” or CDRs. The CDRs from the twochains of each heavy chain/light chain pair mentioned above typicallyare aligned by the framework regions to form a structure that bindsspecifically with a specific epitope on the target protein (e.g.,c-fms). From N-terminal to C-terminal, naturally-occurring light andheavy chain variable regions both typically conform with the followingorder of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Anumbering system has been devised for assigning numbers to amino acidsthat occupy positions in each of these domains. This numbering system isdefined in Kabat Sequences of Proteins of Immunological Interest (1987and 1991, NIH, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883.

The various heavy chain and light chain variable regions provided hereinare depicted in TABLE 2. Each of these variable regions may be attachedto the above heavy and light chain constant regions to form a completeantibody heavy and light chain, respectively. Further, each of the sogenerated heavy and light chain sequences may be combined to form acomplete antibody structure. It should be understood that the heavychain and light chain variable regions provided herein can also beattached to other constant domains having different sequences than theexemplary sequences listed above.

Specific examples of some of the full length light and heavy chains ofthe antibodies that are provided and their corresponding amino acidsequences are summarized in TABLE 1.

TABLE 1 Exemplary Heavy and Light Chains SEQ ID Reference DesignationNO. Amino Acid Sequence H1 109 H1  4QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYYMHWVRQAPGQ 1N1G1GLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGGYSGYDLGYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK H1 13 H2  5QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGGYYWSWIRQPP 1N1G1GKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAAGIAATGTLFDCWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK H1 131 H3  6QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQ 1N1G1GLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDRGQLWLWYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK H1 134 H4  7QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK 1N1G1GLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSSWSYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK H1 143 H5  8EVQLVESGGGLVKPGGSLRLSCAASGFTVSNAWMSWVRQAPGK 1N1G1GLEWVGRIKSKTDGGTTDNAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGGSLLWTGPNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK H1 144 H6  9EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGK 1N1G1GLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEYYGSGGVWYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK H1 16 H7 10EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGK 1N1G1GLEWVGRIKSKTDGWTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDLRITGTTYYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK H1 2 H8 11QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQ 1N1G1GLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARESWFGEVFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK H1 26 H9 12EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGK 1N1G1GLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEYYGSGGVWYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK H1 27 H10 13EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGK 1N1G1GLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDGATVVTPGYYYYGTDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK H1 30 H11 14QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQ 1N1G1GLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARESWFGEVFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK H1 33-1 H12 15QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ 1N1G1GLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAFYYCARDSNWYHNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK H1 33 H13 16QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ 1N1G1GLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAFYYCARDSNWYHNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK H1 34 H14 17EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGK 1N1G1GLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDGATVVTPGYYYYGTDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK H1 39 H15 18EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGK 1N1G1GLEWVGRIKSKTDGGTADYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEGPYSDYGYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK H1 42 H16 19QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQ 1N1G1GLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARESWFGEVFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK H1 64 H17 20EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQATGK 1N1G1GLEWVSGIGTAGDTYYPGSVKGRFNISRENAKNSLYLQMNSLRAGDTAVYYCAREGSWYGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK H1 66 H18 21QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK 1N1G1GLEWVAVIWYDGSNEYYADSVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAHSSGNYYDMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK H1 72 H19 22EVQLVESGGGLVEPGGSLRLSCAASGFTFSTAWMSWVRQAPGK 1N1G1GLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKNEDTAVYYCTTEGPYSNYGYYYYGVDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK H2 103 H20 23EVQLVESGGGLVKPGGSLTLSCAASGFTFNNAWMSWVRQAPGK 1N1G2GLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEYYHILTGSFYYSYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK H1 90 H21 24QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK 1N1G1GLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSSSNFYDMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK H2 131 H22 25QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQSAGKG 1N1G2LEWIGRIYTSGSTHYNPSLKSRIIMSVDTSKNQFSLKLSSVTAADTAVYYCARDRVFYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H2 291 H23 26QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGK 1N1G2GLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGDYSDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK H2 360 H24 27QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGK 1N1G2GLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTVYMELSSLRSEDTAVYYCATGVMITFGGVIVGHSYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK H2 360 H25 28QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGK 1N1G2 SMGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGVMITFGGVIVGHSYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK H2 369 H26 29QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGK 1N1G2GLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATRAGTTLAYYYYAMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK H2 380 H27 30QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKG 1N1G2LEWIGYIYYSGNTNYNPSLKSRFTLSIDTSKNQFSLRLSSVTAADTAVYYCACIATRPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK H2 475 H28 31QVQLVESGGGVVQPGRSLRLSCAASGFTFISYGMHWVRQAPGK 1N1G2GLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCADSSGDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK H2 508 H29 32QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGK 1N1G2GLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATAGLEIRWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK H2 534 H30 33QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPG 1N1G2KGLEWIGYISYSGDTYYNPSLKSRLTISVDTSKHQFSLRLSSVTSADTAVYYCASLDLYGDYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK H2 550 H31 34QVQLVQSGAEVKKPGASVKVSCKASGYTLTSYGISWVRQAPGQ 1N1G2GLEWMGWISAYNGNPNYAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDQGLLGFGELEGLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK H2 65 H32 35EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGK 1N1G2GLEWVGRIKTKTDGGTTDYAAPVKGRFTISRDDSQNTLYLQMNSLKTEDTAVYYCTTEYYGIVTGSFYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK H1 109 L1 36DIQMTQSPSSLSASVGDRVTITCQASQNISNFLDWYQQKPGKAPN 1N1KLLIYDASDLDPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYVSLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 109 L2 37DIQMTQSPSSLSASVGDRVTITCQASQDISNFLDWYQQKPGKAPK 1N1K SMLLIYDASDLDPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYVSLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 131 L3 38DNVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKP 1N1KGQPPQLLIYEASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 134 L4 39DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAP 1N1KKLLIYDASNLEIGVPSRFSGSGSGTDFIFTISSLQPEDIATYYCQQYDNFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 143 L5 40DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAP 1N1KKLLIYDTSNLEPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 144 L6 41DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQHKPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 16 L7 42DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK 1N1KFLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 2 1N1K L8 43DIVMTQSPDSLAVSLGERATINCKSSQSVLDSSDNKNYLAWYQQKPGQPPKLLIYWASNRESGVPDRFSGSGSGTDFSLTISSLQAEDVAVYYCQQYYSDPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 2 1N1K L9 44DIVMTQSPDSLAVSLGERATINCKSSQSVLDSSDNKNYLAWYQQK SMPGQPPKLLIYWASNRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSDPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 27 L10 45DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 30 L11 46DIVMTQSPDSLAVSLGERATIDCKSSQGVLDSSNNKNFLAWYQQ 1N1KKPGQPPKLLIYWASNRESGVPVRFSGSGSGTDFTLTISSLQAEDVALYYCQQYYSDPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 33-1 L12 47DIQMTQSPSSLSASVGDRVTITCRASQSISDYLNWYQQKPGKAPN 1N1KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQTYSDPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 34 L13 48DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 39 L14 49DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK 1N1KVLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 42 L15 50DIVMTQSPDSLAVSLGERATIDCKSSQSVLDSSNNKNFLAWYQQK 1N1KPGQPPKLLIYWASNRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSDPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 64 L16 51EIVLTQSPGTLSLSPGERATLSCRASQSVSSGYLAYLAWYQQKPG 1N1KQAPRLLIYGASSTATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 66 L17 52DIQMTQSPSSLSASVGDRVTITCQASQDISNFLNWYQQRPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 72 L18 53DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQYDNLLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 90 L19 54DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQRYDDLPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 103 L20 55DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQRPGKAP 1N1KKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 131 L21 56DIQMTQSPSSLSASVGDRVTITCRASQGFSNYLAWYQQKPGKVP 1N1KKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 360 L22 57DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWYQQKPGKVP 1N1KQLLIYVASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSGPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 360 L23 58DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWYQQKPGKVPK 1N1K SMLLIYVASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSGPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 369 L24 59DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPN 1N1KLLIHAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPPSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 380 L25 60DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLDWYQQKPGKAP 1N1KKRLIYAASSLQSGVPSRFSGSGSGTEFTLTINSLQPEDFATYYCLQYNSYPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 475 L26 61DIQMIQSPSSLSASVGDRVTITCQASHDISNYLNWYQQKPGKAPK 1N1KFLISDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 508 L27 62DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP 1N1KGQSPQFLIYLGSIRASGVPDRFSGSGSGTDFALTISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 534 L28 63EIVLTQSPDFQSVTPKEKVTITCRASQYIGSSLHWYQQTPDQSPKL 1N1KLINYVSQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSSLPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 550 L29 64DIVMTQSPDSLAVSLGARATISCKSSQSVLYSSNNKNYLAWYQQK 1N1KPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISTLQAEDVAVYYCQQYYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H2 65 L30 65DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAP 1N1KKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDDLLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 13 L31 66DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFIISSLQPEDIATYYCQQFDNLPPTFGGGTKVESKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 26 L32 67DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQHKPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC H1 13H1 13 L33 68DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQR 1NVK2KKPGQSPRRLIYKVSNWDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRGLFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C H1 26H1 26 L34 69DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQR 1NVK2KKPGQSPRRLIYKVSNWDSGVPDRFNGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPITFGQGTGLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Again, each of the exemplary heavy chains (H1, H2, H3 etc.) listed inTABLE 1 can be combined with any of the exemplary light chains shown inTABLE 1 to form an antibody. Examples of such combinations include H1combined with any of L1 through L34; H2 combined with any of L1 throughL34; H3 combined with any of L1 through L34, and so on. In someinstances, the antibodies include at least one heavy chain and one lightchain from those listed in TABLE 1. In some instances, the antibodiescomprise two different heavy chains and two different light chainslisted in TABLE 1. In other instances, the antibodies contain twoidentical light chains and two identical heavy chains. As an example, anantibody or immunologically functional fragment may include two H1 heavychains and two L1 light chains, or two H2 heavy chains and two L2 lightchains, or two H3 heavy chains and two L3 light chains and other similarcombinations of pairs of light chains and pairs of heavy chains aslisted in TABLE 1.

Other antigen binding proteins that are provided are variants ofantibodies formed by combination of the heavy and light chains shown inTABLE 1 and comprise light and/or heavy chains that each have at least70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to the amino acidsequences of these chains. In some instances, such antibodies include atleast one heavy chain and one light chain, whereas in other instancesthe variant forms contain two identical light chains and two identicalheavy chains.

Variable Domains of Antibodies

Also provided are antigen binding proteins that contain an antibodyheavy chain variable region selected from the group consisting ofV_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9,V_(H)10, V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17,V_(H)18, V_(H)19, V_(H)20, V_(H)21, V_(H)22, V_(H)23, V_(H)24, V_(H)25,V_(H)26, V_(H)27, V_(H)28, V_(H)29, V_(H)30, V_(H)31, and V_(H)32,and/or an antibody light chain variable region selected from the groupconsisting of V_(L)1, V_(L)2, V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7,V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13, V_(L)14, V_(L)15,V_(L)16, V_(L)17, V_(L)18, V_(L)19, V_(L)20, V_(L)21, V_(L)22, V_(L)23,V_(L)24, V_(L)25, V_(L)26, V_(L)27, V_(L)28, V_(L)29, V_(L)30, V_(L)31,V_(L)32, V_(L)33, and V_(L)34, as shown in TABLE 2 below, andimmunologically functional fragments, derivatives, muteins and variantsof these light chain and heavy chain variable regions.

Sequence alignments of the various heavy and light chain variableregions, respectively, are provided in FIGS. 1A and 1B.

Antigen binding proteins of this type can generally be designated by theformula “V_(H)x/V_(L)y,” where “x” corresponds to the number of heavychain variable regions and “y” corresponds to the number of the lightchain variable regions (in general, x and y are each 1 or 2) as listedin TABLE 2:

TABLE 2 Exemplary V_(H) and V_(L) Chains Reference DesignationSEQ ID NO. Amino Acid Sequence H1 109 V_(H)1 70QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYYMHVVVRQAPGQGL 1N1G1EWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGGYSGYDLGYYYGMDVWGQGTTVTVSS H1 13 V_(H)2 71QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGGYYWSWIRQPPGKG 1N1G1LEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAAGIAATGTLFDCWGQGTLVTVSS H1 131 V_(H)3 72QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLE 1N1G1WMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDRGQLWLWYYYYYGMDVWGQGTTVTVSS H1 134 V_(H)4 73QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 1N1G1WVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSSWSYYGMDVWGQGTTVTVSS H1 143 V_(H)5 74EVQLVESGGGLVKPGGSLRLSCAASGFTVSNAWMSWVRQAPGKGLE 1N1G1WVGRIKSKTDGGTTDNAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGGSLLWTGPNYYYYGMDVWGQGTTVTVSS H1 144 V_(H)6 75EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 1N1G1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEYYGSGGVWYYGMDVWGQGTTVTVSS H1 16 V_(H)7 76EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 1N1G1WVGRIKSKTDGWTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDLRITGTTYYYYYYGMDVWGQGTTVTVSS H1 2 V_(H)8 77QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLE 1N1G1WMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARESWFGEVFFDYWGQGTLVTVSS H1 26 V_(H)9 78EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 1N1G1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEYYGSGGVWYYGMDVWGQGTTVTVSS H1 27 V_(H)10 79EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 1N1G1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDGATVVTPGYYYYGTDVWGQGTTVTVSS H1 30 V_(H)11 80QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLE 1N1G1WMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARESWFGEVFFDYWGQGTLVTVSS H1 33-1 V_(H)12 81QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHVVVRQAPGQGL 1N1G1EWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAFYYCARDSNVVYHNWFDPWGQGTLVTVSS H1 33 V_(H)13 82QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHVVVRQAPGQGL 1N1G1EWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAFYYCARDSNVVYHNWFDPWGQGTLVTVSS H1 34 V_(H)14 83EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 1N1G1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDGATVVTPGYYYYGTDVWGQGTTVTVSS H1 39 V_(H)15 84EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 1N1G1WVGRIKSKTDGGTADYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEGPYSDYGYYYYGMDVWGQGTTVTVSS H1 42 V_(H)16 85QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLE 1N1G1WMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARESWFGEVFFDYWGQGTLVTVSS H1 64 V_(H)17 86EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQATGKGLE 1N1G1WVSGIGTAGDTYYPGSVKGRFNISRENAKNSLYLQMNSLRAGDTAV YYCAREGSWYGFDYWGQGTLVTVSSH1 66 V_(H)18 87 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 1N1G1WVAVIWYDGSNEYYADSVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAHSSGNYYDMDVWGQGTTVTVSS H1 72 V_(H)19 88EVQLVESGGGLVEPGGSLRLSCAASGFTFSTAWMSWVRQAPGKGLE 1N1G1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKNEDTAVYYCTTEGPYSNYGYYYYGVDVWGQGTTVTVSS H2 103 V_(H)20 89EVQLVESGGGLVKPGGSLTLSCAASGFTFNNAWMSWVRQAPGKGLE 1N1G2WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTEYYHILTGSFYYSYYGMDVWGQGTTVTVSS H1 90 V_(H)21 90QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 1N1G1WVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSSSNFYDMDVWGQGTTVTVSS H2 131 V_(H)22 91QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQSAGKGLE 1N1G2WIGRIYTSGSTHYNPSLKSRIIMSVDTSKNQFSLKLSSVTAADTAV YYCARDRVFYGMDVWGQGTTVTVSSH2 291 V_(H)23 92 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 1N1G2WVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGDYSDYYGMDVWGQGTTVTVSS H2 360 V_(H)24 93QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLE 1N1G2WMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTVYMELSSLRSEDTAVYYCATGVMITFGGVIVGHSYYGMDVWGQGTTVTVSS H2 360 V_(H)25 94QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLE 1N1G2 SMWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGVMITFGGVIVGHSYYGMDVWGQGTTVTVSS H2 369 V_(H)26 95QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLE 1N1G2WMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATRAGTTLAYYYYAMDVWGQGTTVTVSS H2 380 V_(H)27 96QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLE 1N1G2WIGYIYYSGNTNYNPSLKSRFTLSIDTSKNQFSLRLSSVTAADTAV YYCACIATRPFDYWGQGTLVTVSSH2 475 V_(H)28 97 QVQLVESGGGVVQPGRSLRLSCAASGFTFISYGMHWVRQAPGKGLE 1N1G2WVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCADSSGDYYGMDVWGQGTTVTVSS H2 508 V_(H)29 98QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLE 1N1G2WMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATAGLEIRWFDPWGQGTLVTVSS H2 534 V_(H)30 99QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKG 1N1G2LEWIGYISYSGDTYYNPSLKSRLTISVDTSKHQFSLRLSSVTSADTAVYYCASLDLYGDYFDYWGQGTLVTVSS H2 550 V_(H)31 100QVQLVQSGAEVKKPGASVKVSCKASGYTLTSYGISWVRQAPGQGLE 1N1G2WMGWISAYNGNPNYAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDQGLLGFGELEGLFDYWGQGTLVTVSS H2 65 V_(H)32 101EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 1N1G2WVGRIKTKTDGGTTDYAAPVKGRFTISRDDSQNTLYLQMNSLKTEDTAVYYCTTEYYGIVTGSFYYYYYGMDVWGQGTTVTVSS H1 109 V_(L)1 102DIQMTQSPSSLSASVGDRVTITCQASQNISNFLDWYQQKPGKAPNL 1N1KLIYDASDLDPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYV SLPLTFGGGTKVEIK H1 109V_(L)2 103 DIQMTQSPSSLSASVGDRVTITCQASQDISNFLDWYQQKPGKAPKL 1N1K SMLIYDASDLDPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYV SLPLTFGGGTKVEIK H1 131V_(L)3 104 DNVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPG 1N1KQPPQLLIYEASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQSIQLPLTFGGGTKVEIKH1 134 V_(L)4 105 DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAPKL 1N1KLIYDASNLEIGVPSRFSGSGSGTDFIFTISSLQPEDIATYYCQQYD NFPFTFGGGTKVEIK H1 143V_(L)5 106 DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAPKL 1N1KLIYDTSNLEPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLLTFGQGTRLEIK H1 144V_(L)6 107 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQHKPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLLTFGGGTKVEIK H1 16V_(L)7 108 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKF 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLITFGQGTRLEIK H1 2 1N1KV_(L)8 109 DIVMTQSPDSLAVSLGERATINCKSSQSVLDSSDNKNYLAVVYQQKPGQPPKLLIYWASNRESGVPDRFSGSGSGTDFSLTISSLQAEDVAV YYCQQYYSDPFTFGPGTKVDIKH1 2 1N1K V_(L)9 110 DIVMTQSPDSLAVSLGERATINCKSSQSVLDSSDNKNYLAVVYQQK SMPGQPPKLLIYWASNRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCQQYYSDPFTFGPGTKVDIKH1 27 V_(L)10 111 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLLTFGGGTKVEIK H1 30V_(L)11 112 DIVMTQSPDSLAVSLGERATIDCKSSQGVLDSSNNKNFLAWYQQKP 1N1KGQPPKLLIYWASNRESGVPVRFSGSGSGTDFTLTISSLQAEDVALY YCQQYYSDPFTFGPGTKVDIKH1 33-1 V_(L)12 113 DIQMTQSPSSLSASVGDRVTITCRASQSISDYLNWYQQKPGKAPNL 1N1KLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQTY SDPFTFGPGTKVDIK H1 34V_(L)13 114 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLLTFGGGTKVEIK H1 39V_(L)14 115 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKV 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLLTFGGGTKVEIK H1 42V_(L)15 116 DIVMTQSPDSLAVSLGERATIDCKSSQSVLDSSNNKNFLAWYQQKP 1N1KGQPPKLLIYWASNRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQYYSDPFTFGPGTKVDIKH1 64 V_(L)16 117 EIVLTQSPGTLSLSPGERATLSCRASQSVSSGYLAYLAWYQQKPGQ 1N1KAPRLLIYGASSTATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPITFGQGTRLEIK H1 66V_(L)17 118 DIQMTQSPSSLSASVGDRVTITCQASQDISNFLNWYQQRPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLPFTFGPGTKVDIK H1 72V_(L)18 119 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQYD NLLTFGGGTKVEIK H1 90V_(L)19 120 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQRYD DLPITFGQGTRLEIK H2 103V_(L)20 121 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQRPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLLTFGGGTKVEIK H2 131V_(L)21 122 DIQMTQSPSSLSASVGDRVTITCRASQGFSNYLAVVYQQKPGKVPK 1N1KLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKY NSAPLTFGGGTKVEIK H2 360V_(L)22 123 DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWYQQKPGKVPQL 1N1KLIYVASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYN SGPFTFGPGTKVDIK H2 360V_(L)23 124 DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWYQQKPGKVPKL 1N1K SMLIYVASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYN SGPFTFGPGTKVDIK H2 369V_(L)24 125 DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPNL 1N1KLIHAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY ITPPSFGQGTKLEIK H2 380V_(L)25 126 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLDWYQQKPGKAPKR 1N1KLIYAASSLQSGVPSRFSGSGSGTEFTLTINSLQPEDFATYYCLQYN SYPITFGQGTRLEIK H2 475V_(L)26 127 DIQMIQSPSSLSASVGDRVTITCQASHDISNYLNWYQQKPGKAPKF 1N1KLISDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD NLPLTFGGGTKVEIK H2 508V_(L)27 128 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 1N1KQSPQFLIYLGSIRASGVPDRFSGSGSGTDFALTISRVEAEDVGVYY CMQALQTPRTFGQGTKVEIKH2 534 V_(L)28 129 EIVLTQSPDFQSVTPKEKVTITCRASQYIGSSLHWYQQTPDQSPKL 1N1KLINYVSQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQSS SLPFTFGPGTKVDIK H2 550V_(L)29 130 DIVMTQSPDSLAVSLGARATISCKSSQSVLYSSNNKNYLAWYQQKP 1N1KGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISTLQAEDVAVY YCQQYYTTPPTFGQGTKVEIKH2 65 V_(L)30 131 DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYD DLLTFGGGTKVEIK H1 13V_(L)31 132 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKL 1N1KLIYDASNLETGVPSRFSGSGSGTDFTFIISSLQPEDIATYYCQQFD NLPPTFGGGTKVESK H1 26V_(L)32 133 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNVVYQHKPGKAPK 1N1KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQY DNLLTFGGGTKVEIKH1 13H1 13 V_(L)33 134 DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRP1NVK2KK GQSPRRLIYKVSNWDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRGLFTFGPGTKVDIK H1 26H1 26 V_(L)34 135DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRP 1NVK2KKGQSPRRLIYKVSNWDSGVPDRFNGSGSGTDFTLKISRVEAEDVGV YYCMQGTHWPITFGQGTGLEIK

Each of the heavy chain variable regions listed in TABLE 2 may becombined with any of the light chain variable regions shown in TABLE 2to form an antigen binding protein. Examples of such combinationsinclude V_(H)1 combined with any of V_(L)1, V_(L)2, V_(L)3, V_(L)4,V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12,V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17, V_(L)18, V_(L)19, V_(L)20,V_(L)21, V_(L)22, V_(L)23, V_(L)24, V_(L)25, V_(L)26, V_(L)27, V_(L)28,V_(L)29, V_(L)30, V_(L)31, V_(L)32, V_(L)33, or V_(L)34; V_(H)2 combinedwith any of V_(L)1, V_(L)2, V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7,V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13, V_(L)14, V_(L)15,V_(L)16, V_(L)17, V_(L)18, V_(L)19, V_(L)20, V_(L)21, V_(L)22, V_(L)23,V_(L)24, V_(L)25, V_(L)26, V_(L)27, V_(L)28, V_(L)29, or V_(L)30, orV_(H)3 combined with any of V_(L)1, V_(L)2, V_(L)3, V_(L)4, V_(L)5,V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13,V_(L)14, V_(L)15, V_(L)16, V_(L)17, V_(L)18, V_(L)19, V_(L)20, V_(L)21,V_(L)22, V_(L)23, V_(L)24, V_(L)25, V_(L)26, V_(L)27, V_(L)28, V_(L)29,V_(L)30, V_(L)31, V_(L)32, V_(L)33, or V_(L)34, and so on.

In some instances, the antigen binding protein includes at least oneheavy chain variable region and/or one light chain variable region fromthose listed in TABLE 2. In some instances, the antigen binding proteinincludes at least two different heavy chain variable regions and/orlight chain variable regions from those listed in TABLE 2. An example ofsuch an antigen binding protein comprises (a) one V_(H)1, and (b) one ofV_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10,V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17, V_(H)18,V_(H)19, V_(H)20, V_(H)21, V_(H)22, V_(H)23, V_(H)24, V_(H)25, V_(H)26,V_(H)27, V_(H)28, V_(H)29, V_(H)30, V_(H)31, or V_(H)32. Another examplecomprises (a) one V_(H)2, and (b) one of V_(H)1, V_(H)3, V_(H)4, V_(H)5,V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10, V_(H)11, V_(H)12, V_(H)13,V_(H)14, V_(H)15, V_(H)16, V_(H)17, V_(H)18, V_(H)19, V_(H)20, V_(H)21,V_(H)22, V_(H)23, V_(H)24, V_(H)25, V_(H)26, V_(H)27, V_(H)28, V_(H)29,V_(H)30, V_(H)31, or V_(H)32. Again another example comprises (a) oneV_(H)3, and (b) one of V_(H)1, V_(H)2, V_(H)4, V_(H)5, V_(H)6, V_(H)7,V_(H)8, V_(H)9, V_(H)10, V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15,V_(H)16, V_(H)17, V_(H)18, V_(H)19, V_(H)20, V_(H)21, V_(H)22, V_(H)23,V_(H)24, V_(H)25, V_(H)26, V_(H)27, V_(H)28, V_(H)29, V_(H)30, V_(H)31,or V_(H)32 etc.

Again another example of such an antigen binding protein comprises (a)one V_(L)1, and (b) one of V_(L)2, V_(L)3, V_(L)4, V_(L)5, V_(L)6,V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13, V_(L)14,V_(L)15, V_(L)16, V_(L)17, V_(L)18, V_(L)19, V_(L)20, V_(L)21, V_(L)22,V_(L)23, V_(L)24, V_(L)25, V_(L)26, V_(L)27, V_(L)28, V_(L)29, V_(L)30,V_(L)31, V_(L)32, V_(L)33, or V_(L)34. Again another example of such anantigen binding protein comprises (a) one V_(L)2, and (b) one of V_(L)1,V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10,V_(L)11, V_(L)12, V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17, V_(L)18,V_(L)19, V_(L)20, V_(L)21, V_(L)22, V_(L)23, V_(L)24, V_(L)25, V_(L)26,V_(L)27, V_(L)28, V_(L)29, V_(L)30, V_(L)31, V_(L)32, V_(L)33, andV_(L)34. Again another example of such an antigen binding proteincomprises (a) one V_(L)3, and (b) one of V_(L)1, V_(L)2, V_(L)4, V_(L)5,V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13,V_(L)14, V_(L)15, V_(L)16, V_(L)17, V_(L)18, V_(L)19, V_(L)20, V_(L)21,V_(L)22, V_(L)23, V_(L)24, V_(L)25, V_(L)26, V_(L)27, V_(L)28, V_(L)29,V_(L)30, V_(L)31, V_(L)32, V_(L)33, or V_(L)34, etc.

The various combinations of heavy chain variable regions may be combinedwith any of the various combinations of light chain variable regions.

In other instances, the antigen binding protein contains two identicallight chain variable regions and/or two identical heavy chain variableregions. As an example, the antigen binding protein may be an antibodyor immunologically functional fragment that includes two light chainvariable regions and two heavy chain variable regions in combinations ofpairs of light chain variable regions and pairs of heavy chain variableregions as listed in TABLE 2.

Some antigen binding proteins that are provided comprise a heavy chainvariable domain comprising a sequence of amino acids that differs fromthe sequence of a heavy chain variable domain selected from V_(H)1,V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10,V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17, V_(H)18,V_(H)19, V_(H)20, V_(H)21, V_(H)22, V_(H)23, V_(H)24, V_(H)25, V_(H)26,V_(H)27, V_(H)28, V_(H)29, V_(H)30, V_(H)31, and V_(H)32 at only 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues,wherein each such sequence difference is independently either adeletion, insertion or substitution of one amino acid, with thedeletions, insertions and/or substitutions resulting in no more than 15amino acid changes relative to the foregoing variable domain sequences.The heavy chain variable region in some antigen binding proteinscomprises a sequence of amino acids that has at least 70%, 75%, 80%,85%, 90%, 95%, 97% or 99% sequence identity to the amino acid sequencesof the heavy chain variable region of V_(H)1, V_(H)2, V_(H)3, V_(H)4,V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10, V_(H)11, V_(H)12,V_(H)13, V_(H)14, V_(H)15, V_(H)16, V_(H)17, V_(H)18, V_(H)19, V_(H)20,V_(H)21, V_(H)22, V_(H)23, V_(H)24, V_(H)25, V_(H)26, V_(H)27, V_(H)28,V_(H)29, V_(H)30, V_(H)31, and V_(H)32.

Certain antigen binding proteins comprise a light chain variable domaincomprising a sequence of amino acids that differs from the sequence of alight chain variable domain selected from V_(L)1, V_(L)2, V_(L)3,V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11,V_(L)12, V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17, V_(L)18, V_(L)19,V_(L)20, V_(L)21, V_(L)22, V_(L)23, V_(L)24, V_(L)25, V_(L)26, V_(L)27,V_(L)28, V_(L)29, V_(L)30, V_(L)31, V_(L)32, V_(L)33, or V_(L)34 at only1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues,wherein each such sequence difference is independently either adeletion, insertion or substitution of one amino acid, with thedeletions, insertions and/or substitutions resulting in no more than 15amino acid changes relative to the foregoing variable domain sequences.The light chain variable region in some antigen binding proteinscomprises a sequence of amino acids that has at least 70%, 75%, 80%,85%, 90%, 95%, 97% or 99% sequence identity to the amino acid sequencesof the light chain variable region of V_(L)1, V_(L)2, V_(L)3, V_(L)4,V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12,V_(L)13, V_(L)14, V_(L)15, V_(L)16, V_(L)17, V_(L)18, V_(L)19, V_(L)20,V_(L)21, V_(L)22, V_(L)23, V_(L)24, V_(L)25, V_(L)26, V_(L)27, V_(L)28,V_(L)29, V_(L)30, V_(L)31, V_(L)32, V_(L)33, or V_(L)34.

Still other antigen binding proteins, e.g., antibodies orimmunologically functional fragments, include variant forms of a variantheavy chain and a variant light chain as just described.

CDRs

The antigen binding proteins disclosed herein are polypeptides intowhich one or more CDRs are grafted, inserted and/or joined. An antigenbinding protein can have 1, 2, 3, 4, 5 or 6 CDRs. An antigen bindingprotein thus can have, for example, one heavy chain CDR1 (“CDRH1”),and/or one heavy chain CDR2 (“CDRH2”), and/or one heavy chain CDR3(“CDRH3”), and/or one light chain CDR1 (“CDRL1”), and/or one light chainCDR2 (“CDRL2”), and/or one light chain CDR3 (“CDRL3”). Some antigenbinding proteins include both a CDRH3 and a CDRL3. Specific heavy andlight chain CDRs are identified in TABLES 3A and 3B, respectively.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. in Sequences of Proteins of Immunological Interest, 5th Ed., USDept. of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, 1991. Certain antibodies that are disclosed herein comprise oneor more amino acid sequences that are identical or have substantialsequence identity to the amino acid sequences of one or more of the CDRspresented in TABLE 3A (CDRHs) and TABLE 3B (CDRLs).

TABLE 3A Exemplary CDRH Sequences Contained in Reference DesignationAmino Acid Sequence/SEQ ID NO. H1 13 1N1G1; H2 534 1NG2 CDRH 1-1 SGGYYWS[SEQ ID NO: 136] H1 26 1N1G1; H1 143 1N1G1; CDRH 1-2 NAWMSH1 144 1N1G1; H1 39 1N1G1; [SEQ ID NO: 137] H2 103 1N1G2; H2 65 1N1G2;H1 16 1N1G1; H1 34 1N1G1;H1 27 1N1G1 H1 72 1N1G1 CDRH 1-3 TAWMS[SEQ ID NO: 138] H1 33 1N1G1; H1 33 1 1N1G1 CDRH 1-4 GYYMH[SEQ ID NO: 139] H1 109 1N1G1 CDRH 1-5 AYYMH [SEQ ID NO: 140] H1 4 1N1G1CDRH 1-6 SYDMH [SEQ ID NO: 141] H2 369 1N1G2; H2 508 1N1G2; CDRH 1-7ELSMH H2 360 1N1G2 [SEQ ID NO: 142] H2 475 1N1G2; H1 66 1N1G1; CDRH 1-8SYGMH H1 90 1N1G1; H1 134 1N1G1; [SEQ ID NO: 143] H2 291 1N1G2H2 380 1n1g2 CDRH 1-9 SYYWS [SEQ ID NO: 144] H2 131 1N1G2 CDRH 1-10NYYWS [SEQ ID NO: 145] H1 131 1N1G1 CDRH 1-11 GYYIH [SEQ ID NO: 146]H2 550 1N1G2; H1 2 1N1G1; H1 CDRH 1-12 SYGIS 30 1N1G1; H1 42 1N1G1[SEQ ID NO: 147] H1 13 1N1G1 CDRH 2-1 YIYYSGSTNYNPSLKS [SEQ ID NO: 148]H2 534 1N1G2 CDRH 2-2 YISYSGDTYYNPSLKS [SEQ ID NO: 149]H1 26 1N1G1; H1 144 1N1G1; CDRH 2-3 RIKSKTDGGTTDYAAPVKGH2 103 1N1G2; H1 72 1N1G1; [SEQ ID NO: 150] H1 34 1N1G1; H1 27 1N1G1H1 143 1N1G1 CDRH 2-4 RIKSKTDGGTTDNAAPVKG [SEQ ID NO: 151] H1 39 1N1G1CDRH 2-5 RIKSKTDGGTADYAAPVKG [SEQ ID NO: 152] H2 65 1N1G2 CDRH 2-6RIKTKTDGGTTDYAAPVKG [SEQ ID NO: 153] H1 16 1N1G1 CDRH 2-7RIKSKTDGWTTDYAAPVKG [SEQ ID NO: 154] H1 33 1N1G1; H1 33 1 1N1G1;CDRH 2-8 WINPNSGGTNYAQKFQG H1 109 1N1G1; H1 131 1N1G1 [SEQ ID NO: 155]H1 4 1N1G1 CDRH 2-9 GIGTAGDTYYPGSVKG [SEQ ID NO: 156]H2 369 1N1G2; H2 508 1N1G2; CDRH 2-10 GFDPEDGETIYAQKFQG H2 360 1N1G2[SEQ ID NO: 157] H2 475 1N1G2; H1 90 1N1G1; CDRH 2-11 VIWYDGSNKYYADSVKGH1 134 1N1G1 [SEQ ID NO: 158] H2 380 1N1G2 CDRH 2-12 YIYYSGNTNYNPSLKS[SEQ ID NO: 159] H1 66 1N1G1 CDRH 2-13 VIWYDGSNEYYADSVKG[SEQ ID NO: 160] H2 131 1N1G2 CDRH 2-14 RIYTSGSTHYNPSLKS[SEQ ID NO: 161] H2 291 1N1G2 CDRH 2-15 VIWYDGSYKYYADSVKG[SEQ ID NO: 162] H1 2 1N1G1; H1 30 1N1G1; H1 CDRH 2-16 WISAYNGNTNYAQKLQG42 1N1G1 [SEQ ID NO: 163] H2 550 1N1G2 CDRH 2-17 WISAYNGNPNYAQKFQG[SEQ ID NO: 164] H1 13 1N1G1 CDRH 3-1 GIAATGTLFDC [SEQ ID NO: 165]H1 26 1N1G1; H1 144 1N1G1 CDRH 3-2 EYYGSGGVWYYGMDV [SEQ ID NO: 166]H1 143 1N1G1 CDRH 3-3 GGSLLWTGPNYYYYGMDV [SEQ ID NO: 167]H1 33 1N1G1; H1 33 1 1N1G1 CDRH 3-4 DSNWYHNWFDP [SEQ ID NO: 168]H1 109 1N1G1 CDRH 3-5 GGYSGYDLGYYYGMDV [SEQ ID NO: 169] H1 39 1N1G1CDRH 3-6 EGPYSDYGYYYYGMDV [SEQ ID NO: 170] H1 534 1N1G1 CDRH 3-7LDLYGDYFDY [SEQ ID NO: 171] H1 4 1N1G1 CDRH 3-8 EGSWYGFDY[SEQ ID NO: 172] H1 103 1N1G1 CDRH 3-9 EYYHILTGSFYYSYYGMDV[SEQ ID NO: 173] H2 65 1N1G2 CDRH 3-10 EYYGIVTGSFYYYYYGMDV[SEQ ID NO: 174] H2 369 1N1G2 CDRH 3-11 RAGTTLAYYYYAMDV [SEQ ID NO: 175]H2 508 1N1G2 CDRH 3-12 AGLEIRWFDP [SEQ ID NO: 176] H2 475 1N1G2CDRH 3-13 SSGDYYGMDV [SEQ ID NO: 177] H2 380 1N1 G2 CDRH 3-14 IATRPFDY[SEQ ID NO: 178] H2 131 1N1G2 CDRH 3-15 DRVFYGMDV [SEQ ID NO: 179]H2 291 1N1G2 CDRH 3-16 EGDYSDYYGMDV [SEQ ID NO: 180] H1 131 1N1G1CDRH 3-17 DRGQLWLWYYYYYGMDV [SEQ ID NO: 181] H1 66 1N1G1 CDRH 3-18SSGNYYDMDV [SEQ ID NO: 182] H1 90 1N1G1 CDRH 3-19 SSSNFYDMDV[SEQ ID NO: 183] H1 16 1N1G1 CDRH 3-20 DLRITGTTYYYYYYGMDV[SEQ ID NO: 184] H2 550 1N1G2 CDRH 3-21 DQGLLGFGELEGLFDY[SEQ ID NO: 185] H1 2 1N1G1; H1 30 1N1G1; H1 CDRH 3-22 ESWFGEVFFDY42 1N1G1 [SEQ ID NO: 186] H2 360 1N1G2 CDRH 3-23 GVMITFGGVIVGHSYYGMDV[SEQ ID NO: 187] H1 72 1N1G1 CDRH 3-24 EGPYSNYGYYYYGVDV [SEQ ID NO: 188]H1 34 1N1G1; H1 27 1N1G1 CDRH 3-25 DGATVVTPGYYYYGTDV [SEQ ID NO: 189]H1 134 1N1G1 CDRH 3-26 SSWSYYGMDV [SEQ ID NO: 190]

TABLE 3B Exemplary CDRL Sequences Contained in Reference DesignationAmino Acid Sequence/SEQ ID NO. H1 26H1 26 1VK2KK; H1 13H1 CDRL1-1RSSQSLVYSDGNTYLN 13 1NVK2KK [SEQ ID NO: 191] H1 33 1 1N1K CDRL1-2RASQSISDYLN [SEQ ID NO: 192] H1 2 1N1K CDRL1-3 KSSQSVLDSSDNKNYLA[SEQ ID NO: 193] H1 42 1N1K CDRL1-4 KSSQSVLDSSNNKNFLA [SEQ ID NO: 194]H1 30 1N1K CDRL1-5 KSSQGVLDSSNNKNFLA [SEQ ID NO: 195] H2 369 1N1KCDRL1-6 RASQSISRYLN [SEQ ID NO: 196] H1 131 1N1K CDRL1-7 KSSQSLLSDGKTYLY[SEQ ID NO: 197] H1 16 1N1K; H1 90 1N1K; H1 CDRL1-8 QASQDISNYLN34 1N1K; H1 72 1N1K; H2 103 [SEQ ID NO: 198] 1N1K; H1 27 1N1K; H1 1441N1K; H1 39 1N1K; H1 13 1N1K; H1 26 1N1K H1 143 1N1K; H2 65 1N1K; H1CDRL1-9 QASQDINNYLN 134 1N1K [SEQ ID NO: 199] H2 475 1N1K CDRL1-10QASHDISNYLN [SEQ ID NO: 200] H1 109 1N1K CDRL1-11 QASQNISNFLD[SEQ ID NO: 201] H1 109 1N1K SM CDRL1-12 QASQDISNFLD [SEQ ID NO: 202]H1 66 1N1K CDRL1-13 QASQDISNFLN [SEQ ID NO: 203] H2 550 1N1K CDRL1-14KSSQSVLYSSNNKNYLA [SEQ ID NO: 204] H2 131 1N1K CDRL1-15 RASQGFSNYLA[SEQ ID NO: 205] H2 360 1N1K CDRL1-16 RASQGINNYLA [SEQ ID NO: 206]H2 508 1N1K CDRL1-17 RSSQSLLHSNGYNYLD [SEQ ID NO: 207] H2 534 1N1KCDRL1-18 RASQYIGSSLH [SEQ ID NO: 208] H1 64 1N1K CDRL1-19RASQSVSSGYLAYLA [SEQ ID NO: 209] H2 380 1N1K CDRL1-20 RASQGIRNDLD[SEQ ID NO: 210] H1 26H1 26 1NVK2KK; H1 CDRL2-1 KVSNWDS 13H1 13 1NVK2KK[SEQ ID NO: 211] H1 33 1 1N1K; H2 369 1N1K; CDRL2-2 AASSLQS H2 380 1N1K[SEQ ID NO: 212] H2 550 1N1K CDRL2-3 WASTRES [SEQ ID NO: 213]H1 2 1N1K; H1 42 1N1K; H1 CDRL2-4 WASNRES 30 1N1K [SEQ ID NO: 214]H1 131 1N1K CDRL2-5 EASNRFS [SEQ ID NO: 215] H1 16 1N1K; H1 90 1N1K; H1CDRL2-6 DASNLET 34 1N1K; H2 65 1N1K; H1 72 [SEQ ID NO: 216]1N1K; H2475 1N1K; H2 103 1N1K; H1 27 1N1K; H1 1441N1K; H1 39 1N1K; H1 13 1N1K; H1 26 1N1K; H1 66 1N1K H1 143 1N1K CDRL2-7DTSNLEP [SEQ ID NO: 217] H1 109 1N1K CDRL2-8 DASDLDP [SEQ ID NO: 218]H1 134 1N1K CDRL2-9 DASNLEI [SEQ ID NO: 219] H2 131 1N1K CDRL2-10AASTLQS [SEQ ID NO: 220] H2 360 1N1K CDRL2-11 VASTLQS [SEQ ID NO: 221]H2 534 1N1K CDRL2-12 YVSQSFS [SEQ ID NO: 222] H1 64 1N1K CDRL2-13GASSTAT [SEQ ID NO: 223] h2 508 1NIK CDRL2-14 LGSIRAS [SEQ ID NO: 224]H1 26H1 26 1NVK2KK CDRL3-1 MQGTHWPIT [SEQ ID NO: 225] H1 13H1 13 1NVK2KKCDRL3-2 MQGTHWPRGLFT [SEQ ID NO: 226] H1 33 1 1N1K CDRL3-3 QQTYSDPFT[SEQ ID NO: 227] H1 2 1N1K; H1 42 1N1K; H1 CDRL3-4 QQYYSDPFT 30 1N1K[SEQ ID NO: 228] H2 369 1N1K CDRL3-5 QQSYITPPS [SEQ ID NO: 229]H1 131 1N1K CDRL3-6 MQSIQLPLT [SEQ ID NO: 230] H1 16 1N1K CDRL3-7QQYDNLIT [SEQ ID NO: 231] H1 90 1N1K CDRL3-8 QRYDDLPIT [SEQ ID NO: 232]H1 143 1N1K; H1 34 1N1K; H1 CDRL3-9 QQYDNLLT 72 1N1K; H2 103 1N1K; H1 27[SEQ ID NO: 233] 1N1K; H1 144 1N1K; H1 39 1N1K; H1 26 1N1K H2 65 1N1KCDRL3-10 QQYDDLLT [SEQ ID NO: 234] H2 475 1N1K CDRL3-11 QQYDNLPLT[SEQ ID NO: 235] H2 109 1N1K CDRL3-12 QQYVSLPLT [SEQ ID NO: 236]H1 134 1N1K CDRL3-13 QQYDNFPFT [SEQ ID NO: 237] H1 13 1N1K CDRL3-14QQFDNLPPT [SEQ ID NO: 238] H2 550 1N1K CDRL3-15 QQYYTTPPT[SEQ ID NO: 239] H1 66 1N1K CDRL3-16 QQYDNLPFT [SEQ ID NO: 240]H2 131 1N1K CDRL3-17 QKYNSAPLT [SEQ ID NO: 241] H2 360 1N1K CDRL3-18QKYNSGPFT [SEQ ID NO: 242] H2 508 1N1K CDRL3-19 MQALQTPRT[SEQ ID NO: 243] H2 534 1N1K CDRL3-20 HQSSSLPFT [SEQ ID NO: 244]H1 64 1N1K CDRL3-21 QQYGSSPIT [SEQ ID NO: 245] H2 380 1N1K CDRL3-22LQYNSYPIT [SEQ ID NO: 246]

The structure and properties of CDRs within a naturally occurringantibody has been described, supra. Briefly, in a traditional antibody,the CDRs are embedded within a framework in the heavy and light chainvariable region where they constitute the regions responsible forantigen binding and recognition. A variable region comprises at leastthree heavy or light chain CDRs, see, supra (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, Public Health ServiceN.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342: 877-883), within aframework region (designated framework regions 1-4, FR1, FR2, FR3, andFR4, by Kabat et al., 1991, supra; see also Chothia and Lesk, 1987,supra). The CDRs provided herein, however, may not only be used todefine the antigen binding domain of a traditional antibody structure,but may be embedded in a variety of other polypeptide structures, asdescribed herein.

In one aspect, the CDRs provided are (a) a CDRH selected from the groupconsisting of (i) a CDRH1 selected from the group consisting of SEQ IDNO:136-147; (ii) a CDRH2 selected from the group consisting of SEQ IDNO:148-164; (iii) a CDRH3 selected from the group consisting of SEQ IDNO:165-190; and (iv) a CDRH of (i), (ii) and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more thanfive, four, three, two, or one amino acids; (B) a CDRL selected from thegroup consisting of (i) a CDRL1 selected from the group consisting ofSEQ ID NO:191-210; (ii) a CDRL2 selected from the group consisting ofSEQ ID NO:211-224; (iii) a CDRL3 selected from the group consisting ofSEQ ID NO:225-246; and (iv) a CDRL of (i), (ii) and (iii) that containsone or more amino acid substitutions, deletions or insertions of no morethan five, four, three, two, or one amino acids amino acids.

In another aspect, an antigen binding protein includes 1, 2, 3, 4, 5, or6 variant forms of the CDRs listed in TABLES 3A and 3B, each having atleast 80%, 85%, 90% or 95% sequence identity to a CDR sequence listed inTABLES 3A and 3B. Some antigen binding proteins include 1, 2, 3, 4, 5, 6of the CDRs listed in TABLES 3A and 3B, each differing by no more than1, 2, 3, 4 or 5 amino acids from the CDRs listed in these tables.

In yet another aspect, the CDRs disclosed herein include consensussequences derived from groups of related monoclonal antibodies. Asdescribed herein, a “consensus sequence” refers to amino acid sequenceshaving conserved amino acids common among a number of sequences andvariable amino acids that vary within a given amino acid sequences. TheCDR consensus sequences provided include CDRs corresponding to each ofCDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

Consensus sequences were determined using standard phylogenic analysesof the CDRs corresponding to the V_(H) and V_(L) of anti-c-fmsantibodies. The consensus sequences were determined by keeping the CDRscontiguous within the same sequence corresponding to a V_(H) or V_(L).Briefly, amino acid sequences corresponding to the entire variabledomains of either V_(H) or V_(L) were converted to FASTA formatting forease in processing comparative alignments and inferring phylogenies.Next, framework regions of these sequences were replaced with anartificial linker sequence (“GGGAAAGGGAAA” (SEQ ID NO:325)) so thatexamination of the CDRs alone could be performed without introducing anyamino acid position weighting bias due to coincident events (e.g., suchas unrelated antibodies that serendipitously share a common germlineframework heritage) while still keeping CDRs contiguous within the samesequence corresponding to a V_(H) or V_(L). V_(H) or V_(L) sequences ofthis format were then subjected to sequence similarity alignmentinterrogation using a program that employs a standard ClutalW-likealgorithm (see, Thompson et al., 1994, Nucleic Acids Res. 22:4673-4680).A gap creation penalty of 8.0 was employed along with a gap extensionpenalty of 2.0. This program likewise generated phylograms (phylogenictree illustrations) based on sequence similarity alignments using eitherUPGMA (unweighted pair group method using arithmetic averages) orNeighbor-Joining methods (see, Saitou and Nei, 1987, Molecular Biologyand Evolution 4:406-425) to construct and illustrate similarity anddistinction of sequence groups via branch length comparison andgrouping. Both methods produced similar results but UPGMA-derived treeswere ultimately used as the method employs a simpler and moreconservative set of assumptions. UPGMA-derived trees are shown in FIG. 2where similar groups of sequences were defined as having fewer than 15substitutions per 100 residues (see, legend in tree illustrations forscale) amongst individual sequences within the group and were used todefine consensus sequence collections.

As illustrated in FIG. 2, lineage analysis of a variety of the antigenbinding proteins provided herein resulted in three groups of closelyrelated phylogenically clones, designated as Groups A, B, and C.

The consensus sequences of the various CDR regions of Group A are:

a. a CDRH1 of the generic formula GYTX₁TSYGIS (SEQ ID NO:307), whereinX₁ is selected from the group consisting of F and L;

b. a CDRH2 of the generic formula WISAYNGNX₁NYAQKX₂QG (SEQ ID NO:308),wherein X₁ is selected from the group consisting of T and P, and X₂ isselected from the group consisting of L and F;

c. a CDRH3 of the generic formula X₁X₂X₃X₄X₄X₅FGEX₆X₇X₈X₉FDY (SEQ IDNO:309), wherein X₁ is selected from the group consisting of E and D, X₂is selected from the group consisting of S and Q, X₃ is selected fromthe group consisting of G and no amino acid, X₄ is selected from thegroup consisting of L and no amino acid, X₅ is selected from the groupconsisting of W and G, X₆ is selected from the group consisting of V andL, X₇ is selected from the group consisting of E and no amino acid, X₈is selected from the group consisting of G and no amino acid, and X₉ isselected from the group consisting of F and L;

d. a CDRL1 of the generic formula KSSX₁GVLX₂SSX₃NKNX₄LA (SEQ ID NO:310),wherein X₁ is selected from the group consisting of Q and S, X₂ isselected from the group consisting of D and Y, X₃ is selected from thegroup consisting of N and D, and X₄ is selected from the groupconsisting of F and Y;

e. a CDRL2 of the generic formula WASX₁RES (SEQ ID NO:311), wherein X₁is selected from the group consisting of N and T; and

f. a CDRL3 of the generic formula QQYYX₁X₂PX₃T (SEQ ID NO:312), whereinX₁ is selected from the group consisting of S and T, X₂ is selected fromthe group consisting of D and T, and X₃ is selected from the groupconsisting of F and P.

The consensus sequences of the various CDR regions of Group B are:

a. a CDRH1 having the generic formula GFTX₁X₂X₃AWMS (SEQ ID NO:313),wherein X₁ is selected from the group consisting of F and V, X₂ isselected from the group consisting of S and N, and X₃ is selected fromthe group consisting of N and T;

b. a CDRH2 having the generic formula RIKX₁KTDGX₂TX₃DX₄AAPVKG (SEQ IDNO:314), wherein X₁ is selected from the group consisting of S and T, X₂is selected from the group consisting of G and W, X₃ is selected fromthe group consisting of T and A, and X₄ is selected from the groupconsisting of Y and N;

c. a CDRH3 having the generic formulaX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃YYGX₁₄DV (SEQ ID NO:315), wherein X₁ isselected from the group consisting of E, D and G, X₂ is selected fromthe group consisting of Y, L and no amino acid, X₃ is selected from thegroup consisting of Y, R, G and no amino acid, X₄ is selected from thegroup consisting of H, G, S and no amino acid, X₅ is selected from thegroup consisting of I, A, L and no amino acid, X₆ is selected from thegroup consisting of L, V, T, P and no amino acid, X₇ is selected fromthe group consisting of T, V, Y, G, W and no amino acid, X₈ is selectedfrom the group consisting of G, V, S and T, X₉ is selected from thegroup consisting of S, T, D, N and G, X₁₀ is selected from the groupconsisting of G, F, P, and Y, X₁₁ is selected from the group consistingof G, Y and N, X₁₂ is selected from the group consisting of V and Y, X₁₃is selected from the group consisting of W, S and Y, and X₁₄ is selectedfrom the group consisting of M, T and V;

d. a CDRL1 having the generic formula QASQDIX₁NYLN (SEQ ID NO:316),wherein X₁ is selected from the group consisting of S and N;

e. a CDRL2 having the generic formula DX₁SNLEX₂ (SEQ ID NO:317), whereinX₁ is selected from the group consisting of A and T, and X₂ is selectedfrom the group consisting of T and P; and

f. a CDRL3 having the generic formula QQYDX₁LX₂T (SEQ ID NO:318),wherein X₁ is selected from the group consisting of N and D, and X₂ isselected from the group consisting of L and I.

The consensus sequences of the various CDR regions of Group C are:

a. a CDRH1 having the generic formula GFTFX₁SYGMH (SEQ ID NO:319),wherein X₁ is selected from the group consisting of S and I;

b. a CDRH2 having the generic formula VIWYDGSNX₁YYADSVKG (SEQ IDNO:320), wherein X₁ is selected from the group consisting of E and K;

c. a CDRH3 having the generic formula SSX₁X₂X₃YX₄MDV (SEQ ID NO:321),wherein X₁ is selected from the group consisting of G, S and W, X₂ isselected from the group consisting of N, D and S, X₃ is selected fromthe group consisting of Y and F, and X₄ is selected from the groupconsisting of D and G;

d. a CDRL1 having the generic formula QASX₁DIX₂NX₃LN (SEQ ID NO:322),wherein X₁ is selected from the group consisting of Q and H, X₂ isselected from the group consisting of S and N, and X₃ is selected fromthe group consisting of F and Y;

e. a CDRL2 having the generic formula DASNLEX₁ (SEQ ID NO:323), whereinX₁ is selected from the group consisting of T and I; and

f. a CDRL3 having the generic formula QX₁YDX₂X₃PX₄T (SEQ ID NO:324),wherein X₁ is selected from the group consisting of Q and R, X₂ isselected from the group consisting of N and D, X₃ is selected from thegroup consisting of L and F, and X₄ is selected from the groupconsisting of F, L and I.

In some cases the antigen binding protein comprises at least one CDRH1,CDRH2, or CDRH3 having one of the above consensus sequences. In somecases, the antigen binding protein comprises at least one CDRL1, CDRL2,or CDRL3 having one of the above consensus sequences. In other cases,the antigen binding protein comprises at least two CDRHs according tothe above consensus sequences, and/or at least two CDRLs according tothe above consensus sequences. In one aspect, the CDRHs and/or CDRLs arederived from different groups A, B, and C. In other cases, the antigenbinding protein comprises at least two CDRHs from the same group A, B,or C, and/or at least two CDRLs from the same group A, B, or C. In otheraspects, the antigen binding protein comprises a CDRH1, CDRH2, and CDRH3sequence from the same of the above groups A, B, or C, and/or a CDRL1,CDRL2, and CDRL3 sequence from the same of the above groups A, B, or C.

Hence, some antigen binding proteins that are provided include 1, 2, 3,4, 5 or all 6 of the CDRs from the Group A consensus sequences. Thuscertain antigen binding proteins, for instance, include a CDRH1, aCDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 from the Group A consensussequences set forth above. Other antigen binding proteins that areprovided include 1, 2, 3, 4, 5 or all 6 of the CDRs from the Group Bconsensus sequences. Thus certain antigen binding proteins include, forinstance, a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2 and a CDRL3 fromthe Group B consensus sequences set forth above. Still other antigenbinding proteins that are provided include 1, 2, 3, 4, 5 or all 6 of theCDRs from the Group C consensus sequences. Thus certain antigen bindingproteins include, for instance, a CDRH1, a CDRH2, a CDRH3, a CDRL1, aCDRL2 and a CDRL3 from the Group A consensus sequences set forth above.

Exemplary Antigen Binding Proteins

According to one aspect, provided is an isolated antigen-binding proteinthat binds c-fms comprising (A) one or more heavy chain complementarydetermining regions (CDRHs) selected from the group consisting of: (i) aCDRH1 selected from the group consisting of SEQ ID NO:136-147; (ii) aCDRH2 selected from the group consisting of SEQ ID NO:148-164; (iii) aCDRH3 selected from the group consisting of SEQ ID NO:165-190; and (iv)a CDRH of (i), (ii) and (iii) that contains one or more amino acidsubstitutions, deletions or insertions of no more than five, four,three, four, two or one amino acids; (B) one or more light chaincomplementary determining regions (CDRLs) selected from the groupconsisting of: (i) a CDRL1 selected from the group consisting of SEQ IDNO:191-210; (ii) a CDRL2 selected from the group consisting of SEQ IDNO:211-224; (iii) a CDRL3 selected from the group consisting of SEQ IDNO:225-246; and (iv) a CDRL of (i), (ii) and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more thanfive, four, three, four, two or one amino acids; or (C) one or moreheavy chain CDRHs of (A) and one or more light chain CDRLs of (B).

In yet another embodiment, the isolated antigen-binding protein maycomprise (A) a CDRH selected from the group consisting of (i) a CDRH1selected from the group consisting of SEQ ID NO:136-147; (ii) a CDRH2selected from the group consisting of SEQ ID NO:148-164; and (iii) aCDRH3 selected from the group consisting of SEQ ID NO:165-190; (B) aCDRL selected from the group consisting of (i) a CDRL1 selected from thegroup consisting of SEQ ID NO:191-210; (ii) a CDRL2 selected from thegroup consisting of SEQ ID NO:211-224; and (iii) a CDRL3 selected fromthe group consisting of SEQ ID NO:225-246; or (C) one or more heavychain CDRHs of (A) and one or more light chain CDRLs of (B). In oneembodiment, the isolated antigen-binding protein may include (A) a CDRH1of SEQ ID NO:136-147, a CDRH2 of SEQ ID NO:148-164, and a CDRH3 of SEQID NO:165-190, and (B) a CDRL1 of SEQ ID NO:191-210, a CDRL2 of SEQ IDNO:211-224, and a CDRL3 of SEQ ID NO:225-246.

In another embodiment, the variable heavy chain (VH) has at least 70%,75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity with an amino acidsequence selected from the group consisting of SEQ ID NO:70-101, and/orthe variable light chain (VL) has at least 70%, 75%, 80%, 85%, 90%, 95%,97% or 99% sequence identity with an amino acid sequence selected fromthe group consisting of SEQ ID NO:102-135. In a further embodiment, theVH is selected from the group consisting of SEQ ID NO: 70-101, and/orthe VL is selected from the group consisting of SEQ ID NO: 102-135.

In another aspect, also provided is an isolated antigen binding proteinthat specifically binds to an epitope containing the c-fms subdomainsIg-like 1-1 and Ig-like 1-2 of c-fms.

In a further aspect, there is a provision of an isolated antigen-bindingprotein that binds c-fms, the antigen-binding protein including a CDRH3selected from the group consisting of (1) a CDRH3 selected from thegroup consisting of SEQ ID NOs:165-190, (2) a CDRH3 that differs inamino acid sequence from the CDRH3 of (i) by an amino acid addition,deletion or substitution of not more than two amino acids; and (3) aCDRH3 amino acid sequence selected from the group consisting of (a)X₁X₂X₃X₄X₄X₅FGEX₆X₇X₈X₉FDY (SEQ ID NO:309), wherein X₁ is selected fromthe group consisting of E and D, X₂ is selected from the groupconsisting of S and Q, X₃ is selected from the group consisting of G andno amino acid, X₄ is selected from the group consisting of L and noamino acid, X₅ is selected from the group consisting of W and G, X₆ isselected from the group consisting of V and L, X₇ is selected from thegroup consisting of E and no amino acid, X₈ is selected from the groupconsisting of G and no amino acid, and X₉ is selected from the groupconsisting of F and L (CDRH3 consensus sequence derived from abovedescribed phylogenetic Group A); (b)X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃YYGX₁₄DV (SEQ ID NO:315), wherein X₁ isselected from the group consisting of E, D and G, X₂ is selected fromthe group consisting of Y, L and no amino acid, X₃ is selected from thegroup consisting of Y, R, G and no amino acid, X₄ is selected from thegroup consisting of H, G, S and no amino acid, X₅ is selected from thegroup consisting of I, A, L and no amino acid, X₆ is selected from thegroup consisting of L, V, T, P and no amino acid, X₇ is selected fromthe group consisting of T, V, Y, G, W and no amino acid, X₈ is selectedfrom the group consisting of G, V, S and T, X₉ is selected from thegroup consisting of S, T, D, N and G, X₁₀ is selected from the groupconsisting of G, F, P, and Y, X₁₁ is selected from the group consistingof G, Y and N, X₁₂ is selected from the group consisting of V and Y, X₁₃is selected from the group consisting of W, S and Y, and X₁₄ is selectedfrom the group consisting of M, T and V (CDRH3 consensus sequencederived from above described phylogenetic Group B); and (c)SSX₁X₂X₃YX₄MDV (SEQ ID NO:321), wherein X₁ is selected from the groupconsisting of G, S and W, X₂ is selected from the group consisting of N,D and S, X₃ is selected from the group consisting of Y and F, and X₄ isselected from the group consisting of D and G (CDRH3 consensus sequencederived from above described phylogenetic Group C); or (B) a light chaincomplementary determining region (CDRL) selected from the groupconsisting of (1) a CDRL3 selected from the group consisting of SEQ IDNOs:225-246, (2) a CDRL3 that differs in amino acid sequence from theCDRL3 of (i) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (3) a CDRL3 amino acid sequence selectedfrom the group consisting of (a) QQYYX₁X₂PX₃T (SEQ ID NO:312), whereinX₁ is selected from the group consisting of S and T, X₂ is selected fromthe group consisting of D and T, and X₃ is selected from the groupconsisting of F and P (CDRL3 consensus sequence derived from abovedescribed phylogenetic Group A); (b) QQYDX₁LX₂T (SEQ ID NO:318), whereinX₁ is selected from the group consisting of N and D, and X₂ is selectedfrom the group consisting of L and I (CDRL3 consensus sequence derivedfrom above described phylogenetic Group B); and (c) QX₁YDX₂X₃PX₄T (SEQID NO:324), wherein X₁ is selected from the group consisting of Q and R,X₂ is selected from the group consisting of N and D, X₃ is selected fromthe group consisting of L and F, and X₄ is selected from the groupconsisting of F, L and I (CDRL3 consensus sequence derived from abovedescribed phylogenetic Group C).

In one embodiment, the antigen binding protein that binds c-fmscomprises a CDRH3 according to a consensus sequence of groups A, B, orC, and/or a CDRL3 according to a consensus sequence of groups A, B, orC, and a CDRH1 and/or CDRH2 of any the above groups, and/or a CDRL1and/or a CDRL2 of any of the above groups.

In one embodiment, the isolated antigen binding protein that binds c-fmscomprises a CDRH3 and/or a CDRL3 of Group A, see, supra, and a CDRselected from the group consisting of:

(1) a CDRH1 selected from the group consisting of (a) a CDRH1 of SEQ IDNOs:136-147; (b) a CDRH1 that differs in amino acid sequence from theCDRH1 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRH1 amino acid sequence selectedfrom the group consisting of GYTX₁TSYGIS (SEQ ID NO:307), wherein X₁ isselected from the group consisting of F and L;

(2) CDRH2 selected from the group consisting of (a) a CDRH2 of SEQ IDNOs:148-164; (b) a CDRH2 that differs in amino acid sequence from theCDRH2 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRH2 amino acid sequence selectedfrom the group consisting of WISAYNGNX₁NYAQKX₂QG (SEQ ID NO:308),wherein X₁ is selected from the group consisting of T and P, and X₂ isselected from the group consisting of L and F;

(3) a CDRL1 selected from the group consisting of (a) a CDRL1 of SEQ IDNOs:191-210; (b) a CDRL1 that differs in amino acid sequence from theCDRL1 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRL1 amino acid sequence selectedfrom the group consisting of KSSX₁GVLX₂SSX₃NKNX₄LA (SEQ ID NO:310),wherein X₁ is selected from the group consisting of Q and S, X₂ isselected from the group consisting of D and Y, X₃ is selected from thegroup consisting of N and D, and X₄ is selected from the groupconsisting of F and Y; and

(4) a CDRL2 selected from the group consisting of: (a) a CDRL2 of SEQ IDNOs:211-224; (b) a CDRL2 that differs in amino acid sequence from theCDRL2 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRL2 amino acid sequence selectedfrom the group consisting of WASX₁RES (SEQ ID NO:311), wherein X₁ isselected from the group consisting of N and T.

In one embodiment, the isolated antigen binding protein that binds c-fmscomprises a CDRH3 and/or a CDRL3 of Group B, see, supra, and a CDRselected from the group consisting of:

(1) a CDRH1 selected from the group consisting of (a) a CDRH1 of SEQ IDNOs:136-147; (b) a CDRH1 that differs in amino acid sequence from theCDRH1 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRH1 amino acid sequence selectedfrom the group consisting of GFTX₁X₂X₃AWMS (SEQ ID NO:313), wherein X₁is selected from the group consisting of F and V, X₂ is selected fromthe group consisting of S and N, and X₃ is selected from the groupconsisting of N and T;

(2) a CDRH2 selected from the group consisting of (a) a CDRH2 of SEQ IDNOs:148-164; (b) a CDRH2 that differs in amino acid sequence from theCDRH2 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRH2 amino acid sequence selectedfrom the group consisting of RIKX₁KTDGX₂TX₃DX₄AAPVKG (SEQ ID NO:314),wherein X₁ is selected from the group consisting of S and T, X₂ isselected from the group consisting of G and W, X₃ is selected from thegroup consisting of T and A, and X₄ is selected from the groupconsisting of Y and N;

(3) a CDRL1 selected from the group consisting of (a) a CDRL1 of SEQ IDNOs:191-210; (b) a CDRL1 that differs in amino acid sequence from theCDRL1 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRL1 amino acid sequence selectedfrom the group consisting of QASQDIX₁NYLN (SEQ ID NO:316), wherein X₁ isselected from the group consisting of S and N; and

(4) a CDRL2 selected from the group consisting of (a) a CDRL2 of SEQ IDNOs:211-224; (b) a CDRL2 that differs in amino acid sequence from theCDRL2 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRL2 amino acid sequence selectedfrom the group consisting of DX₁SNLEX₂ (SEQ ID NO:317), wherein X₁ isselected from the group consisting of A and T, and X₂ is selected fromthe group consisting of T and P.

In one embodiment, the isolated antigen binding protein that binds c-fmscomprises a CDRH3 and a CDRL3 of Group C, see, supra, and a CDR selectedfrom the group consisting of

(1) a CDRH1 selected from the group consisting of (a) a CDRH1 of SEQ IDNOs:136-147; (b) a CDRH1 that differs in amino acid sequence from theCDRH1 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRH1 amino acid sequence selectedfrom the group consisting of GFTFX₁SYGMH (SEQ ID NO:319), wherein X₁ isselected from the group consisting of S and I;

(2) a CDRH2 selected from the group consisting of (a) a CDRH2 of SEQ IDNOs:148-164; (b) a CDRH2 that differs in amino acid sequence from theCDRH2 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRH2 amino acid sequence selectedfrom the group consisting of VIWYDGSNX₁YYADSVKG (SEQ ID NO:320), whereinX₁ is selected from the group consisting of E and K;

(3) a CDRL1 selected from the group consisting of (a) a CDRL1 of SEQ IDNOs:191-210; (b) a CDRL1 that differs in amino acid sequence from theCDRL1 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRL1 amino acid sequence selectedfrom the group consisting of QASX₁DIX₂NX₃LN (SEQ ID NO:322), wherein X₁is selected from the group consisting of Q and H, X₂ is selected fromthe group consisting of S and N, and X₃ is selected from the groupconsisting of F and Y;

(4) a CDRL2 selected from the group consisting of (a) a CDRL2 of SEQ IDNOs:211-224; (b) a CDRL2 that differs in amino acid sequence from theCDRL2 of (a) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (c) a CDRL2 amino acid sequence selectedfrom the group consisting of DASNLEX₁ (SEQ ID NO:323), wherein X₁ isselected from the group consisting of T and I.

In yet another embodiment, the isolated antigen binding proteindescribed hereinabove comprises a first amino acid sequence comprisingat least one of the above CDRH consensus sequences, and a second aminoacid sequence comprising at least one of the above CDRL consensussequences. In one aspect, the first amino acid sequence comprises atleast two of the above CDRH consensus sequences, and/or the second aminoacid sequence comprises at least two of the above consensus sequences.In again another aspect, the first amino acid sequence comprises atleast two CDRHs of the same of the above groups A, B, or C, and/or thesecond amino acid sequence comprises at least two CDRLs of the same ofthe above groups A, B, or C. In yet other aspects, the first and secondamino acid sequences comprise at least one CDRH and one CDRL,respectively, of the same of the above groups A, B, or C. In yet afurther aspect, the first amino acid sequence comprises a CDRH1, aCDRH2, and a CDRH3 of the same of the above groups A, B, or C, and/orthe second amino acid sequence comprises a CDRL1, a CDRL2, and a CDRL3of the same of the above groups A, B, or C.

In certain embodiments, the first and the second amino acid sequence arecovalently bonded to each other.

In a further embodiment, the first amino acid sequence of the isolatedantigen-binding protein includes the CDRH3 of SEQ ID NO:165-190, CDRH2of SEQ ID NO:148-164, and CDRH1 of SEQ ID NO:136-147, and/or the secondamino acid sequence of the isolated antigen binding protein comprisesthe CDRL3 of SEQ ID NO:225-246, CDRL2 of SEQ ID NO:211-224, and CDRL1 ofSEQ ID NO:191-210.

In a further embodiment, the antigen binding protein comprises at leasttwo CDRH sequences of heavy chain sequences H1, H2, H3, H4, H5, H6, H7,H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21, H22,H23, H24, H25, H26, H27, H28, H29, H30, H31, or H32, as shown in TABLE4A. In again a further embodiment, the antigen binding protein comprisesat least two CDRL sequences of light chain sequences L1, L2, L3, L4, L5,L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, L17, L18, L19, L20,L21, L22, L23, L24, L25, L26, L27, L28, L29, L30, L31, L32, L33, or L34,as shown in TABLE 4B. In again a further embodiment, the antigen bindingprotein comprises at least two CDRH sequences of heavy chain sequencesH1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16,H17, H18, H19, H20, H21, H22, H23, H24, H25, H26, H27, H28, H29, H30,H31, or H32, as shown in TABLE 4A, and at least two CDRLs of light chainsequences L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14,L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L25, L26, L27, L28,L29, L30, L31, L32, L33, or L34, as shown in TABLE 4B.

In again another embodiment, the antigen binding protein comprises theCDRH1, CDRH2, and CDRH3 sequences of heavy chain sequences H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18,H19, H20, H21, H22, H23, H24, H25, H26, H27, H28, H29, H30, H31, or H32,as shown in TABLE 4A. In yet another embodiment, the antigen bindingprotein comprises the CDRL1, CDRL2, and CDRL3 sequences of light chainsequences L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14,L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L25, L26, L27, L28,L29, L30, L31, L32, L33, or L34, as shown in TABLE 4B.

In yet another embodiment, the antigen binding protein comprises all sixCDRs of H1 and L1, or H1 and L2, or H2 and L31, or H2 and L33, or H3 andL3, or H4 and L4, or H5 and L5, or H6 and L6, or H7 and L7, or H8 andL8, or H8 and L9, or H9 and L32, or H9 and L34, or H10 and L10, or H11and L11, or H12 and L12, or H13 and L12, or H14 and L13, or H15 and L14,or H16 and L15, or H17 and L16, or H18 and L17, or H19 and L18, or H20and L20, or H21 and L19, or H22 and L21, or H24 and L22, or H25 and L23,or H26 and L24, or H27 and L25, or H28 and L26, or H29 and L27, or H30and L28, or H31 and L29, or H32 and L30, as shown in TABLES 4A and 4B.

The sequence information for specific antibodies prepared and identifiedas described in the Examples below are summarized in Table 4C. For easeof reference, in some instances an abbreviated form of the referencenumber is used herein in which the last number of the reference isdropped. Thus, for instance, 1.109.1 is sometime simply referred to as1.109; 1.109.1 SM is referred to as 1.109 SM; 1.2.1 is referred to as1.2; 1.2.1 SM is referred to as 1.2 SM; 2.360.1 is referred to as 2.360,2.360.1 SM is referred to as 2.360 SM; etc.

TABLE 4A Full Variable Full Heavy Variable Heavy CDRH1 CDRH2 CDRH3 HeavySEQ Heavy SEQ CDRH1 SEQ CDRH2 SEQ CDRH3 SEQ Reference (H#) ID NO (VH#)ID NO (CDRH1-#) ID NO (CDRH2-#) ID NO (CDRH3-#) ID NO H1 109 1N1G1 H1 4V_(H)1 70 CDRH 1-5 140 CDRH 2-8 155 CDRH 3-5 169 H1 13 1N1G1 H2 5 V_(H)271 CDRH 1-1 136 CDRH 2-1 148 CDRH 3-1 165 H1 131 1N1G1 H3 6 V_(H)3 72CDRH 1-11 146 CDRH 2-8 155 CDRH 3-17 181 H1 134 1N1G1 H4 7 V_(H)4 73CDRH 1-8 143 CDRH 2-11 158 CDRH 3-26 190 H1 143 1N1G1 H5 8 V_(H)5 74CDRH 1-2 137 CDRH 2-4 151 CDRH 3-3 167 H1 144 1N1G1 H6 9 V_(H)6 75 CDRH1-2 137 CDRH 2-3 150 CDRH 3-2 166 H1 16 1N1G1 H7 10 V_(H)7 76 CDRH 1-2137 CDRH 2-7 154 CDRH 3-20 184 H1 2 1N1G1 H8 11 V_(H)8 77 CDRH 1-12 147CDRH 2-16 163 CDRH 3-22 186 H1 26 1N1G1 H9 12 V_(H)9 78 CDRH 1-2 137CDRH 2-3 150 CDRH 3-2 166 H1 27 1N1G1 H10 13 V_(H)10 79 CDRH 1-2 137CDRH 2-3 150 CDRH 3-25 189 H1 30 1N1G1 H11 14 V_(H)11 80 CDRH 1-12 147CDRH 2-16 163 CDRH 3-22 186 H1 33-1 1N1G1 H12 15 V_(H)12 81 CDRH 1-4 139CDRH 2-8 155 CDRH 3-4 168 H1 33 1N1G1 H13 16 V_(H)13 82 CDRH 1-4 139CDRH 2-8 155 CDRH 3-4 168 H1 34 1N1G1 H14 17 V_(H)14 83 CDRH 1-2 137CDRH 2-3 150 CDRH 3-25 189 H1 39 1N1G1 H15 18 V_(H)15 84 CDRH 1-2 137CDRH 2-5 152 CDRH 3-6 170 H1 42 1N1G1 H16 19 V_(H)16 85 CDRH 1-12 147CDRH 2-16 163 CDRH 3-22 186 H1 64 1N1G1 H17 20 V_(H)17 86 CDRH 1-6 141CDRH 2-9 156 CDRH 3-8 172 H1 66 1N1G1 H18 21 V_(H)18 87 CDRH 1-8 143CDRH 2-13 160 CDRH 3-18 182 H1 72 1N1G1 H19 22 V_(H)19 88 CDRH 1-3 138CDRH 2-3 150 CDRH 3-24 188 H2 103 1N1G2 H20 23 V_(H)20 89 CDRH 1-2 137CDRH 2-3 150 CDRH 3-9 173 H1 90 1N1G1 H21 24 V_(H)21 90 CDRH 1-8 143CDRH 2-11 158 CDRH 3-19 183 H2 131 1N1G2 H22 25 V_(H)22 91 CDRH 1-10 145CDRH 2-14 161 CDRH 3-15 179 H2 291 1N1G2 H23 26 V_(H)23 92 CDRH 1-8 143CDRH 2-15 162 CDRH 3-16 180 H2 360 1N1G2 H24 27 V_(H)24 93 CDRH 1-7 142CDRH 2-10 157 CDRH 3-23 187 H2 360 1N1G2SM H25 28 V_(H)25 94 CDRH 1-7142 CDRH 2-10 157 CDRH 3-23 187 H2 369 1N1G2 H26 29 V_(H)26 95 CDRH 1-7142 CDRH 2-10 157 CDRH 3-11 175 H2 380 1N1G2 H27 30 V_(H)27 96 CDRH 1-9144 CDRH 2-12 159 CDRH 3-14 178 H2 475 1N1G2 H28 31 V_(H)28 97 CDRH 1-8143 CDRH 2-11 158 CDRH 3-13 177 H2 508 1N1G2 H29 32 V_(H)29 98 CDRH 1-7142 CDRH 2-10 157 CDRH 3-12 176 H2 534 1N1G2 H30 33 V_(H)30 99 CDRH 1-1136 CDRH 2-2 149 CDRH 3-7 171 H2 550 1N1G2 H31 34 V_(H)31 100 CDRH 1-12147 CDRH 2-17 164 CDRH 3-21 185 H2 65 1N1G2 H32 35 V_(H)32 101 CDRH 1-2137 CDRH 2-6 153 CDRH 3-10 174

TABLE 4B Full Variable Full Light Variable Light CDRL1 CDRL2 CDRL3 LightSEQ Light SEQ CDRL1 SEQ CDRL2 SEQ CDRL3 SEQ Reference (L#) ID NO (VL#)ID NO (CDRL1-#) ID NO (CDRL2-#) ID NO (CDRL3-#) ID NO H1 109 1N1K L1 36V_(L)1 102 CDRL 1-12 202 CDRL 2-8 218 CDRL3-12 236 H1 109 1N1K SM L2 37V_(L)2 103 CDRL 1-11 201 CDRL 2-8 218 CDRL3-12 236 H1 131 1N1K L3 38V_(L)3 104 CDRL 1-7 197 CDRL 2-5 215 CDRL3-6 230 H1 134 1N1K L4 39V_(L)4 105 CDRL 1-9 199 CDRL 2-9 219 CDRL3-13 237 H1 143 1N1K L5 40V_(L)5 106 CDRL 1-9 199 CDRL 2-7 217 CDRL3-9 233 H1 144 1N1K L6 41V_(L)6 107 CDRL 1-8 198 CDRL 2-6 216 CDRL3-9 233 H1 16 1N1K L7 42 V_(L)7108 CDRL 1-8 198 CDRL 2-6 216 CDRL3-7 231 H1 2 1N1K L8 43 V_(L)8 109CDRL 1-3 193 CDRL 2-4 214 CDRL3-4 228 H1 2 1N1K SM L9 44 V_(L)9 110 CDRL1-3 193 CDRL 2-4 214 CDRL3-4 228 H1 27 1N1K L10 45 V_(L)10 111 CDRL 1-8198 CDRL 2-6 216 CDRL3-9 233 H1 30 1N1K L11 46 V_(L)11 112 CDRL 1-5 195CDRL 2-4 214 CDRL3-4 228 H1 33-1 1N1K L12 47 V_(L)12 113 CDRL 1-2 192CDRL 2-2 212 CDRL3-3 227 H1 34 1N1K L13 48 V_(L)13 114 CDRL 1-8 198 CDRL2-6 216 CDRL3-9 233 H1 39 1N1K L14 49 V_(L)14 115 CDRL 1-8 198 CDRL 2-6216 CDRL3-9 233 H1 42 1N1K L15 50 V_(L)15 116 CDRL 1-4 194 CDRL 2-4 214CDRL3-4 228 H1 64 1N1K L16 51 V_(L)16 117 CDRL 1-19 209 CDRL 2-13 223CDRL3-21 245 H1 66 1N1K L17 52 V_(L)17 118 CDRL 1-13 203 CDRL 2-6 216CDRL3-16 240 H1 72 1N1K L18 53 V_(L)18 119 CDRL 1-8 198 CDRL 2-6 216CDRL3-9 233 H1 90 1N1K L19 54 V_(L)19 120 CDRL 1-8 198 CDRL 2-6 216CDRL3-8 232 H2 103 1N1K L20 55 V_(L)20 121 CDRL 1-8 198 CDRL 2-6 216CDRL3-9 233 H2 131 1N1K L21 56 V_(L)21 122 CDRL 1-15 205 CDRL 2-10 220CDRL3-17 241 H2 360 1N1K L22 57 V_(L)22 123 CDRL 1-16 206 CDRL 2-11 221CDRL3-18 242 H2 360 1N1K SM L23 58 V_(L)23 124 CDRL 1-16 206 CDRL 2-11221 CDRL3-18 242 H2 369 1N1K L24 59 V_(L)24 125 CDRL 1-6 196 CDRL 2-2212 CDRL3-5 229 H2 380 1N1K L25 60 V_(L)25 126 CDRL 1-20 210 CDRL 2-2212 CDRL3-22 246 H2 475 1N1K L26 61 V_(L)26 127 CDRL 1-10 200 CDRL 2-6216 CDRL3-11 235 H2 508 1N1K L27 62 V_(L)27 128 CDRL 1-17 207 CDRL 2-14224 CDRL3-19 243 H2 534 1N1K L28 63 V_(L)28 129 CDRL 1-18 208 CDRL 2-12222 CDRL3-20 244 H2 550 1N1K L29 64 V_(L)29 130 CDRL 1-14 204 CDRL 2-3213 CDRL3-15 239 H2 65 1N1K L30 65 V_(L)30 131 CDRL 1-9 199 CDRL 2-6 216CDRL3-10 234 H1 13 1N1K L31 66 V_(L)31 132 CDRL 1-8 198 CDRL 2-6 216CDRL3-14 238 H1 26 1N1K L32 67 V_(L)32 133 CDRL 1-8 198 CDRL 2-6 216CDRL3-9 233 H1 13H1 13 1NVK2KK L33 68 V_(L)33 134 CDRL 1-1 191 CDRL 2-1211 CDRL3-2 226 H1 26H1 26 1NVK2KK L34 69 V_(L)34 135 CDRL 1-1 191 CDRL2-1 211 CDRL3-1 225

TABLE 4C Full Full Variable Variable Heavy Light Heavy Light CDRH1 CDRH2CDRH3 CDRL1 CDRL2 CDRL3 SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ Ref. No.ID NO ID NO ID NO ID NO ID NO ID NO ID NO ID NO ID NO ID NO 1.109.1 4 3670 102 140 155 169 202 218 236 1.109.1 SM 4 37 70 103 140 155 169 201218 236 1.13.1 5 66 71 132 136 148 165 198 216 238 1.13.13.1 5 68 71 134136 148 165 191 211 226 1.131.1 6 38 72 104 146 155 181 197 215 2301.134.1 7 39 73 105 143 158 190 199 219 237 1.143.1 8 40 74 106 137 151167 199 217 233 1.144.1 9 41 75 107 137 150 166 198 216 233 1.16.1 10 4276 108 137 154 184 198 216 231 1.2.1 11 43 77 109 147 163 186 193 214228 1.2.1 SM 11 44 77 110 147 163 186 193 214 228 1.26.1 12 67 78 133137 150 166 198 216 233 1.26.26.1 12 69 78 135 137 150 166 191 211 2251.27.1 13 45 79 111 137 150 189 198 216 233 1.30.1 14 46 80 112 147 163186 195 214 228 1.33-1.1 15 47 81 113 139 155 168 192 212 227 1.33.1 1647 82 113 139 155 168 192 212 227 1.34.1 17 48 83 114 137 150 189 198216 233 1.39.1 18 49 84 115 137 152 170 198 216 233 1.42.1 19 50 85 116147 163 186 194 214 228 1.64.1 20 51 86 117 141 156 172 209 223 2451.66.1 21 52 87 118 143 160 182 203 216 240 1.72.1 22 53 88 119 138 150188 198 216 233 2.103.1 23 55 89 121 137 150 173 198 216 233 1.90.1 2454 90 120 143 158 183 198 216 232 2.131.1 25 56 91 122 145 161 179 205220 241 2.291.1 26 92 143 162 180 2.360.1 27 57 93 123 142 157 187 206221 242 2.360.1 SM 28 58 94 124 142 157 187 206 221 242 2.369.1 29 59 95125 142 157 175 196 212 229 2.380.1 30 60 96 126 144 159 178 210 212 2462.475.1 31 61 97 127 143 158 177 200 216 235 2.508.1 32 62 98 128 142157 176 207 224 243 2.534.1 33 63 99 129 136 149 171 208 222 244 2.550.134 64 100 130 147 164 185 204 213 239 2.65.1 35 65 101 131 137 153 174199 216 234

In one aspect, the isolated antigen-binding proteins provided herein canbe a monoclonal antibody, a polyclonal antibody, a recombinant antibody,a human antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof.

In another embodiment, the antibody fragment of the isolatedantigen-binding proteins provided herein can be a Fab fragment, a Fab′fragment, an F(ab′)₂ fragment, an Fv fragment, a diabody, or a singlechain antibody molecule.

In a further embodiment, the isolated antigen binding protein providedherein is a human antibody and can be of the IgG1-, IgG2-IgG3- orIgG4-type.

In another embodiment, the antigen binding protein consists of a just alight or a heavy chain polypeptide as set forth in Tables 4A-4C. In someembodiments, the antigen binding protein consists just of a variablelight or variable heavy domain such as those listed in Tables 4A-4C.Such antigen binding proteins can be pegylated with one or more PEGmolecules.

In yet another aspect, the isolated antigen-binding protein providedherein can be coupled to a labeling group and can compete for binding tothe extracellular portion of human c-fms with an antigen binding proteinof one of the isolated antigen-binding proteins provided herein. In oneembodiment, the isolated antigen binding protein provided herein canreduce monocyte chemotaxis, inhibit monocyte migration into tumors orinhibit accumulation and function of tumor associated macrophage in atumor when administered to a patient.

As will be appreciated by those in the art, for any antigen bindingprotein with more than one CDR from the depicted sequences, anycombination of CDRs independently selected from the depicted sequencesis useful. Thus, antigen binding proteins with one, two, three, four,five or six of independently selected CDRs can be generated. However, aswill be appreciated by those in the art, specific embodiments generallyutilize combinations of CDRs that are non-repetitive, e.g., antigenbinding proteins are generally not made with two CDRH2 regions, etc.

Some of the antigen binding proteins provided are discussed in moredetail below.

Antigen Binding Proteins and Binding Epitopes and Binding Domains

When an antigen binding protein is said to bind an epitope withinspecified residues, such as c-fms, or the extracellular domain of c-fms,for example, what is meant is that the antigen binding proteinspecifically binds to a specified portion of c-fms. In some embodiments,the antigen binding protein specifically binds to a polypeptideconsisting of the specified residues (e.g., a specified segment ofc-fms). Such an antigen binding protein typically does not contact everyresidue within c-fms, or the extracellular domain of c-fms. Nor doesevery single amino acid substitution or deletion within c-fms, or theextracellular domain of c-fms, necessarily significantly affect bindingaffinity.

Epitope specificity and the binding domain(s) of an antigen bindingprotein can be determined by a variety of methods. Some methods, forexample, can use truncated portions of an antigen. Other methods utilizeantigen mutated at one or more specific residues.

With respect to methods using truncated portions of an antigen, in oneexemplary approach, a collection of overlapping peptides can be used.The overlapping peptides consist of about 15 amino acids spanning thesequence of the antigen and differing in increments of a small number ofamino acids (e.g., three amino acids). The peptides are immobilizedwithin the wells of a microtiter dish or at different locations on amembrane. Immobilization can be effected by biotinylating one terminusof the peptides. Optionally, different samples of the same peptide canbe biotinylated at the amino- and the carboxy-terminus and immobilizedin separate wells for purposes of comparison. This is useful foridentifying end-specific antigen binding proteins. Optionally,additional peptides can be included terminating at a particular aminoacid of interest. This approach is useful for identifying end-specificantigen binding proteins to internal fragments of c-fms (or theextracellular domain of c-fms). An antigen binding protein orimmunologically functional fragment is screened for specific binding toeach of the various peptides. The epitope is defined as occurring with asegment of amino acids that is common to all peptides to which theantigen binding protein shows specific binding. Details regarding aspecific approach for defining an epitope are set forth in Example 12.

As demonstrated in Example 12, in one embodiment the antigen bindingproteins provided herein are capable of binding a polypeptide thatincludes the Ig-like domain 1 and the Ig-like domain 2 in combination;however, they do not bind a polypeptide containing primarily the Ig-likedomain 1 or primarily the Ig-like domain 2 alone. The binding epitopesof such antigen binding proteins are thus composed three-dimensionallyof the Ig-like 1 and Ig-like 2 domains in combination. As highlighted inFIG. 8, these two domains comprise amino acids 20 through 223 of c-fmsextracellular domain, which has the following amino acid sequence:

(SEQ ID NO: 326) IPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTL VPALVRIRGEAAQIV.

The amino acid sequence used in Example 12 to represent the Ig-like 1domain corresponds to amino acids 20-126 of the sequence depicted inFIG. 8 (i.e., amino acids 20-126 of SEQ ID NO:1, namelyIPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLP). The amino acid sequence usedto represent the Ig-like 2 domain alone corresponded to amino acids85-223 of the sequence depicted in FIG. 8 (i.e., amino acids 85-223 ofSEQ ID NO:1, namelyTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPALVR1RGEAAQIV).

Thus, an antigen binding protein in one embodiment can bind orspecifically bind to a region within a cfms protein (e.g., the maturefull length protein), where the region has the amino acid sequencespecified in SEQ ID NO:326. In some embodiments, the antigen bindingprotein binds or specifically binds to a polypeptide consistingessentially of or consisting of the amino acids residues as set forth inSEQ ID NO:326.

In another embodiment, the antigen binding protein can bind orspecifically bind to a polypeptide consisting of SEQ ID NO:326 but notto a polypeptide consisting of amino acids 20-126 of the sequencedepicted in FIG. 8 (i.e., amino acids 20-126 of SEQ ID NO:1). In anotheraspect, the antigen binding protein can bind or specifically bind to apolypeptide consisting of SEQ ID NO:326 but not to a polypeptideconsisting of amino acids 85-223 of the sequence depicted in FIG. 8(i.e., amino acids 85-223 of SEQ ID NO:1). In yet another embodiment,the antigen binding protein can bind or specifically bind to apolypeptide consisting of SEQ ID NO:326 but not to a polypeptideconsisting of amino acids 20-126 of the sequence depicted in FIG. 8(i.e., amino acids 20-126 of SEQ ID NO:1) or to a polypeptide consistingof amino acids 85-223 of the sequence depicted in FIG. 8 (i.e., aminoacids 85-223 of SEQ ID NO:1).

In another approach, the domain(s)/region(s) containing residues thatare in contact with or are buried by an antibody can be identified bymutating specific residues in an antigen (e.g., a wild-type antigen) anddetermining whether the antigen binding protein can bind the mutatedprotein. By making a number of individual mutations, residues that playa direct role in binding or that are in sufficiently close proximity tothe antibody such that a mutation can affect binding between the antigenbinding protein and antigen can be identified. From a knowledge of theseamino acids, the domain(s) or region(s) of the antigen that containresidues in contact with the antigen binding protein or covered by theantibody can be elucidated. Such a domain typically includes the bindingepitope of an antigen binding protein. One specific example of thisgeneral approach utilizes an arginine/glutamic acid scanning protocol(see, e.g., Nanevicz, T., et al., 1995, J. Biol. Chem., 270:37,21619-21625 and Zupnick, A., et al., 2006, J. Biol. Chem., 281:29,20464-20473). In general, arginine and glutamic acids are substituted(typically individually) for an amino acid in the wild-type polypeptidebecause these amino acids are charged and bulky and thus have thepotential to disrupt binding between an antigen binding protein and anantigen in the region of the antigen where the mutation is introduced.Arginines and lysines that exist in the wild-type antigen are replacedwith glutamic acid. A variety of such individual mutants are obtainedand the collected binding results analyzed to determine what residuesaffect binding.

Example 14 describes arginine/glutamic acid scanning of human c-fms forc-fms binding proteins provided herein. A series of 95 mutant humanc-fms antigens were created, with each mutant antigen having a singlemutation. Binding of each mutant c-fms antigen with selected c-fmsantigen binding proteins provided herein was measured and compared tothe ability of these selected binding proteins to bind wild-type c-fmsantigen (SEQ ID NO:1). A reduction in binding between an antigen bindingprotein and a mutant c-fms antigen as used herein means that there is areduction in binding affinity (e.g., as measured by known methods suchas Biacore testing as described in the examples) and/or a reduction inthe total binding capacity of the antigen binding protein (e.g., asevidenced by a decrease in Bmax in a plot of antigen binding proteinconcentration versus antigen concentration). A significant reduction inbinding indicates that the mutated residue is directly involved inbinding to the antigen binding protein or is in close proximity to thebinding protein when the binding protein is bound to antigen.

In some embodiments, a significant reduction in binding means that thebinding affinity and/or capacity between an antigen binding protein anda mutant c-fms antigen is reduced by greater than 40%, greater than 50%,greater than 55%, greater than 60%, greater than 65%, greater than 70%,greater than 75%, greater than 80%, greater than 85%, greater than 90%or greater than 95% relative to binding between the binding protein anda wild type c-fms antigen (e.g., the extracellular domain shown in SEQID NO:1). In certain embodiments, binding is reduced below detectablelimits. In some embodiments, a significant reduction in binding isevidenced when binding of an antigen binding protein to a mutant c-fmsantigen is less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%,15% or 10%) of the binding observed between the antigen binding proteinand a wild-type c-fms antigen (e.g., the extracellular domain shown inSEQ ID NO:1). Such binding measurements can be made using a variety ofbinding assays known in the art. A specific example of one such assay isdescribed in Example 14.

In some embodiments, antigen binding proteins are provided that exhibitsignificantly lower binding for a mutant c-fms antigen in which aresidue in a wild-type c-fms antigen (e.g., SEQ ID NO:1) is substitutedwith arginine or glutamic acid. In one such embodiment, binding of anantigen binding protein is significantly reduced for a mutant c-fmsantigen having any one or more (e.g., 1, 2, 3 or 4) of the followingmutations: E29R, Q121R, T152R, and K185E as compared to a wild-typec-fms (e.g., SEQ ID NO:1). In the shorthand notation used here, theformat is: Wild type residue: Position in polypeptide: Mutant residue,with the numbering of the residues as indicated in SEQ ID NO:1. In someembodiments, binding of an antigen binding protein is significantlyreduced for a mutant c-fms antigen having any one or more (e.g., 1, 2,3, 4 or 5) of the following mutations: E29R, Q121R, S172R, G274R, andY276R as compared to a wild-type c-fms (e.g., SEQ ID NO:1). In anotherembodiment, an antigen binding protein exhibits significantly lowerbinding for a mutant c-fms antigen containing any one or more (e.g., 1,2, 3, 4, 5 etc. up to 23) of the following mutations: R106E, H151R,T152R, Y154R, S155R, W159R, Q171R, S172R, Q173R, G183R, R184E, K185E,E218R, A220R, S228R, H239R, N240R, K259E, G274R, N275R, Y276R, S277R,and N282R as compared to a wild-type c-fms (e.g., SEQ ID NO:1). In evenmore embodiments, binding of an antigen binding protein is significantlyreduced for a mutant c-fms antigen containing any one or more (e.g., 1,2, 3, 4 or 5) of the following mutations: K102E, R144E, R146E, D174R,and A226R as compared to binding to a wild-type c-fms (e.g., SEQ IDNO:1). In still other embodiments, an antigen binding protein exhibitssignificantly reduced binding for a mutant c-fms antigen containing anyone or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) of the following mutations:W50R, A74R, Y100R, D122R, T130R, G161R, Y175R, and A179R as compared toa wild-type c-fms (e.g., SEQ ID NO:1).

Although the mutant forms just listed are referenced with respect to thewild-type extracellular domain sequence shown in SEQ ID NO:1, it will beappreciated that in an allelic variant of c-fms the amino acid at theindicated position could differ. Antigen binding proteins showingsignificantly lower binding for such allelic forms of c-fms are alsocontemplated. Accordingly, in one embodiment, an antigen binding proteinhas significantly reduced binding for an allelic c-fms antigen ascompared to a wild-type c-fms (e.g., SEQ ID NO:1) where one or more ofthe following residues (e.g., 1, 2, 3 or 4) of the allelic antigen arereplaced with arginine or glutamic acid as indicated: 29R, 121R, 152R,and 185E (Position in polypeptide: Mutant residue, with the numbering ofthe residues as indicated in SEQ ID NO:1). In some embodiments, anantigen binding protein exhibits significantly reduced binding for anallelic c-fms antigen in which one or more (e.g., 1, 2, 3, 4 or 5) ofthe following residues are replaced with arginine or glutamic acid asindicated: 29R, 121R, 172R, 274R, and 276R as compared to its ability tobind a wild-type c-fms (e.g., SEQ ID NO:1). In another embodiment, anantigen binding protein shows significantly reduced binding for anallelic c-fms antigen in which one or more (e.g., 1, 2, 3, 4, 5 etc. upto 23) of the following residues are replaced with arginine or glutamicacid as indicated: 106E, 151R, 152R, 154R, 155R, 159R, 171R, 172R, 173R,183R, 184E, 185E, 218R, 220R, 228R, 239R, 240R, 259E, 274R, 275R, 276R,277R, and 282R as compared to its ability to bind a wild-type c-fms(e.g., SEQ ID NO:1). In even more embodiments, an antigen bindingprotein has significantly reduced binding for an allelic c-fms antigenin which any one or more (e.g., 1, 2, 3, 4 or 5) of the followingresidues are replaced with arginine or glutamic acid as indicated: 102E,144E, 146E, 174R, and 226R as compared to its ability to bind awild-type c-fms (e.g., SEQ ID NO:1). In still other embodiments, anantigen binding protein exhibits significantly reduced binding for anallelic c-fms antigen in which any one or more (e.g., 1, 2, 3, 4, 5, 6,7 or 8) of the following residues are replaced with arginine or glutamicacid as indicated: 50R, 74R, 100R, 122R, 130R, 161R, 175R, and 179R ascompared to its ability to bind a wild-type c-fms (e.g., SEQ ID NO:1).

In some embodiments, binding of an antigen binding protein issignificantly reduced for a mutant c-fms antigen in which the residue ata selected position in the wild-type c-fms antigen is mutated to anyother residue. For instance, in one embodiment, an antigen bindingprotein exhibits significantly reduced binding for a mutant c-fmsantigen containing a single amino acid substitution at one or more(e.g., 1, 2, 3 or 4) of positions 29, 121, 152, and 185 (where thepositions are as indicated in SEQ ID NO:1) as compared to its ability tobind a wild-type c-fms (e.g., SEQ ID NO:1). In some embodiments, anantigen binding protein has significantly reduced binding for a mutantc-fms antigen containing a single amino acid substitution at one or more(e.g., 1, 2, 3, 4 or 5) of positions 29, 121, 172, 274 and 276 of SEQ IDNO:1 as compared to its ability to bind a wild-type c-fms (e.g., SEQ IDNO:1). In another embodiment, binding of an antigen binding protein issignificantly reduced for a mutant c-fms antigen containing a singleamino acid substitution at one or more (e.g., 1, 2, 3, 4, 5 etc. up to23) of positions 106, 151, 152, 154, 155, 159, 171, 172, 173, 183, 184,185, 218, 220, 228, 239, 240, 259, 274, 275, 276, 277, and 282 of SEQ IDNO:1 as compared to binding to a wild-type c-fms of SEQ ID NO:1. In evenmore embodiments, an antigen binding protein has significantly lowerbinding for a mutant c-fms antigen containing a single amino acidsubstitution at one or more (e.g., 1, 2, 3, 4 or 5) of positions 102,144, 146, 174, and 226 of SEQ ID NO:1 as compared to its ability to binda wild-type c-fms (e.g., SEQ ID NO:1). In still other embodiments, anantigen binding protein has significantly lower binding for a mutantc-fms antigen containing a single amino acid substitution at one or more(e.g., 1, 2, 3, 4, 5, 6, 7 or 8) of positions 50, 74, 100, 122, 130,161, 175, and 179 as compared to its ability to bind a wild-type c-fms(e.g., SEQ ID NO:1).

As noted above, residues directly involved in binding or covered by anantigen binding protein can be identified from scanning results. Theseresidues can thus provide an indication of the domains or regions of SEQID NO:1 that contain the binding region(s) to which antigen bindingproteins binds. As can be seen from the results summarized in Example14, in one embodiment an antigen binding protein binds to a domaincontaining amino acids 29-185 of SEQ ID NO:1. In another embodiment, theantigen binding protein binds to a region containing amino acids 29-276of SEQ ID NO:1. In other embodiments, the antigen binding protein bindsto a region containing amino acids 106-282 of SEQ ID NO:1. In stillother embodiments, the antigen binding protein binds to regioncontaining amino acids 102-226 of SEQ ID NO:1. In yet anotherembodiment, the antigen binding protein binds to a region containingamino acids 50-179 of SEQ ID NO: 1. In some embodiments, the antigenbinding proteins binds to the foregoing regions within a fragment of thefull length sequence of SEQ ID NO:1. In other embodiments, antigenbinding proteins bind to polypeptides consisting of these regions. Incertain embodiments, an antigen binding protein binds to a regioncontaining amino acids 90-282 of SEQ ID NO:1. In another embodiment, anantigen binding protein binds to one or both of the following regions:amino acids 90-185 and amino acids 217-282 of SEQ ID NO:1. In yetanother embodiment, an antigen binding protein binds to one or both ofthe following regions: amino acids 121-185 and amino acids 217-277 ofSEQ ID NO:1.

Competing Antigen Binding Proteins

In another aspect, antigen binding proteins are provided that competewith one of the exemplified antibodies or functional fragments bindingto the epitope described above for specific binding to c-fms. Suchantigen binding proteins may also bind to the same epitope as one of theherein exemplified antigen binding proteins, or an overlapping epitope.Antigen binding proteins and fragments that compete with or bind to thesame epitope as the exemplified antigen binding proteins are expected toshow similar functional properties. The exemplified antigen bindingproteins and fragments include those described above, including thosewith the heavy and light chains, variable region domains and CDRsincluded in TABLES 1, 2, 3, and 4A-C. Thus, as a specific example, theantigen binding proteins that are provided include those that competewith an antibody having:

(a) all 6 of the CDRs listed for an antibody listed in Table 4C;

(b) a VH and a VL listed for an antibody listed in Table 4C; or

(c) two light chains and two heavy chains as specified for an antibodylisted in Table 4C.

Monoclonal Antibodies

The antigen binding proteins that are provided include monoclonalantibodies that bind to c-fms. Monoclonal antibodies may be producedusing any technique known in the art, e.g., by immortalizing spleencells harvested from the transgenic animal after completion of theimmunization schedule. The spleen cells can be immortalized using anytechnique known in the art, e.g., by fusing them with myeloma cells toproduce hybridomas. Myeloma cells for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Examples of suitable cell lines foruse in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul;examples of cell lines used in rat fusions include R210. RCY3, Y3-Ag1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions areU-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

In some instances, a hybridoma cell line is produced by immunizing ananimal (e.g., a transgenic animal having human immunoglobulin sequences)with a c-fms immunogen; harvesting spleen cells from the immunizedanimal; fusing the harvested spleen cells to a myeloma cell line,thereby generating hybridoma cells; establishing hybridoma cell linesfrom the hybridoma cells, and identifying a hybridoma cell line thatproduces an antibody that binds a c-fms polypeptide. Such hybridoma celllines, and anti-c-fms monoclonal antibodies produced by them, areaspects of the present application.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any technique known in the art. Hybridomas or mAbs may be furtherscreened to identify mAbs with particular properties, such as theability to block a Wnt induced activity. Examples of such screens areprovided in the examples below.

Chimeric and Humanized Antibodies

Chimeric and humanized antibodies based upon the foregoing sequences arealso provided. Monoclonal antibodies for use as therapeutic agents maybe modified in various ways prior to use. One example is a chimericantibody, which is an antibody composed of protein segments fromdifferent antibodies that are covalently joined to produce functionalimmunoglobulin light or heavy chains or immunologically functionalportions thereof. Generally, a portion of the heavy chain and/or lightchain is identical with or homologous to a corresponding sequence inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is/are identical with or homologous to a corresponding sequencein antibodies derived from another species or belonging to anotherantibody class or subclass. For methods relating to chimeric antibodies,see, for example, U.S. Pat. No. 4,816,567; and Morrison et al., 1985,Proc. Natl. Acad. Sci. USA 81:6851-6855, which are hereby incorporatedby reference. CDR grafting is described, for example, in U.S. Pat. No.6,180,370, No. 5,693,762, No. 5,693,761, No. 5,585,089, and No.5,530,101.

Generally, the goal of making a chimeric antibody is to create a chimerain which the number of amino acids from the intended patient species ismaximized. One example is the “CDR-grafted” antibody, in which theantibody comprises one or more complementarity determining regions(CDRs) from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the antibody chain(s) is/areidentical with or homologous to a corresponding sequence in antibodiesderived from another species or belonging to another antibody class orsubclass. For use in humans, the variable region or selected CDRs from arodent antibody often are grafted into a human antibody, replacing thenaturally-occurring variable regions or CDRs of the human antibody.

One useful type of chimeric antibody is a “humanized” antibody.Generally, a humanized antibody is produced from a monoclonal antibodyraised initially in a non-human animal. Certain amino acid residues inthis monoclonal antibody, typically from non-antigen recognizingportions of the antibody, are modified to be homologous to correspondingresidues in a human antibody of corresponding isotype. Humanization canbe performed, for example, using various methods by substituting atleast a portion of a rodent variable region for the correspondingregions of a human antibody (see, e.g., U.S. Pat. No. 5,585,089, andU.S. Pat. No. 5,693,762; Jones et al., 1986, Nature 321:522-525;Riechmann et al., 1988, Nature 332:323-27; Verhoeyen et al., 1988,Science 239:1534-1536),

In one aspect, the CDRs of the light and heavy chain variable regions ofthe antibodies provided herein (see, TABLE 3) are grafted to frameworkregions (FRs) from antibodies from the same, or a different,phylogenetic species. For example, the CDRs of the heavy and light chainvariable regions V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7,V_(H)8, V_(H)9, V_(H)10, V_(H)11, V_(H)12, V_(H)13, V_(H)14, V_(H)15,V_(H)16, V_(H)17, V_(H)18, V_(H)19, V_(H)20, V_(H)21, V_(H)22, V_(H)23,V_(H)24, V_(H)25, V_(H)26, V_(H)27, V_(H)28, V_(H)29, V_(H)30, V_(H)31,and V_(H)32, and/or V_(L)1, V_(L)2, V_(L)3, V_(L)4, V_(L)5, V_(L)6,V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, V_(L)12, V_(L)13, V_(L)14,V_(L)15, V_(L)16, V_(L)17, V_(L)18, V_(L)19, V_(L)20, V_(L)21, V_(L)22,V_(L)23, V_(L)24, V_(L)25, V_(L)26, V_(L)27, V_(L)28, V_(L)29, V_(L)30,V_(L)31, V_(L)32, V_(L)33 and V_(L)34 can be grafted to consensus humanFRs. To create consensus human FRs, FRs from several human heavy chainor light chain amino acid sequences may be aligned to identify aconsensus amino acid sequence. In other embodiments, the FRs of a heavychain or light chain disclosed herein are replaced with the FRs from adifferent heavy chain or light chain. In one aspect, rare amino acids inthe FRs of the heavy and light chains of anti-c-fms antibody are notreplaced, while the rest of the FR amino acids are replaced. A “rareamino acid” is a specific amino acid that is in a position in which thisparticular amino acid is not usually found in an FR. Alternatively, thegrafted variable regions from the one heavy or light chain may be usedwith a constant region that is different from the constant region ofthat particular heavy or light chain as disclosed herein. In otherembodiments, the grafted variable regions are part of a single chain Fvantibody.

In certain embodiments, constant regions from species other than humancan be used along with the human variable region(s) to produce hybridantibodies.

Fully Human Antibodies

Fully human antibodies are also provided. Methods are available formaking fully human antibodies specific for a given antigen withoutexposing human beings to the antigen (“fully human antibodies”). Onespecific means provided for implementing the production of fully humanantibodies is the “humanization” of the mouse humoral immune system.Introduction of human immunoglobulin (Ig) loci into mice in which theendogenous Ig genes have been inactivated is one means of producingfully human monoclonal antibodies (mAbs) in mouse, an animal that can beimmunized with any desirable antigen. Using fully human antibodies canminimize the immunogenic and allergic responses that can sometimes becaused by administering mouse or mouse-derived mAbs to humans astherapeutic agents.

Fully human antibodies can be produced by immunizing transgenic animals(usually mice) that are capable of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Antigens for this purpose typically have six or more contiguous aminoacids, and optionally are conjugated to a carrier, such as a hapten.See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; andBruggermann et al., 1993, Year in Immunol. 7:33. In one example of sucha method, transgenic animals are produced by incapacitating theendogenous mouse immunoglobulin loci encoding the mouse heavy and lightimmunoglobulin chains therein, and inserting into the mouse genome largefragments of human genome DNA containing loci that encode human heavyand light chain proteins. Partially modified animals, which have lessthan the full complement of human immunoglobulin loci, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies that are immunospecific for the immunogen but havehuman rather than murine amino acid sequences, including the variableregions. For further details of such methods, see, for example,WO96/33735 and WO94/02602. Additional methods relating to transgenicmice for making human antibodies are described in U.S. Pat. No.5,545,807; U.S. Pat. No. 6,713,610; U.S. Pat. No. 6,673,986; U.S. Pat.No. 6,162,963; U.S. Pat. No. 5,545,807; U.S. Pat. No. 6,300,129; U.S.Pat. No. 6,255,458; U.S. Pat. No. 5,877,397; U.S. Pat. No. 5,874,299 andU.S. Pat. No. 5,545,806; in PCT publications WO91/10741, WO90/04036, andin EP 546073B1 and EP 546073A1.

The transgenic mice described above, referred to herein as “HuMab” mice,contain a human immunoglobulin gene minilocus that encodes unrearrangedhuman heavy ([mu] and [gamma]) and [kappa] light chain immunoglobulinsequences, together with targeted mutations that inactivate theendogenous [mu] and [kappa] chain loci (Lonberg et al., 1994, Nature368:856-859). Accordingly, the mice exhibit reduced expression of mouseIgM or [kappa] and in response to immunization, and the introduced humanheavy and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgG [kappa] monoclonalantibodies (Lonberg et al., supra.; Lonberg and Huszar, 1995, Intern.Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci.764:536-546). The preparation of HuMab mice is described in detail inTaylor et al., 1992, Nucleic Acids Research 20:6287-6295; Chen et al.,1993, International Immunology 5:647-656; Tuaillon et al., 1994, J.Immunol. 152:2912-2920; Lonberg et al., 1994, Nature 368:856-859;Lonberg, 1994, Handbook of Exp. Pharmacology 113:49-101; Taylor et al.,1994, International Immunology 6:579-591; Lonberg and Huszar, 1995,Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. N.Y.Acad. Sci. 764:536-546; Fishwild et al., 1996, Nature Biotechnology14:845-851; the foregoing references are hereby incorporated byreference in their entirety for all purposes. See, further U.S. Pat. No.5,545,806; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,625,126; U.S. Pat.No. 5,633,425; U.S. Pat. No. 5,789,650; U.S. Pat. No. 5,877,397; U.S.Pat. No. 5,661,016; U.S. Pat. No. 5,814,318; U.S. Pat. No. 5,874,299;and U.S. Pat. No. 5,770,429; as well as U.S. Pat. No. 5,545,807;International Publication Nos. WO 93/1227; WO 92/22646; and WO 92/03918,the disclosures of all of which are hereby incorporated by reference intheir entirety for all purposes. Technologies utilized for producinghuman antibodies in these transgenic mice are disclosed also in WO98/24893, and Mendez et al., 1997, Nature Genetics 15:146-156, which arehereby incorporated by reference. For example, the HCo7 and HCo12transgenic mice strains can be used to generate anti-c-fms antibodies.Further details regarding the production of human antibodies usingtransgenic mice are provided in the examples below.

Using hybridoma technology, antigen-specific human mAbs with the desiredspecificity can be produced and selected from the transgenic mice suchas those described above. Such antibodies may be cloned and expressedusing a suitable vector and host cell, or the antibodies can beharvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage-display libraries(as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; andMarks et al., 1991, J. Mol. Biol. 222:581). Phage display techniquesmimic immune selection through the display of antibody repertoires onthe surface of filamentous bacteriophage, and subsequent selection ofphage by their binding to an antigen of choice. One such technique isdescribed in PCT Publication No. WO 99/10494 (hereby incorporated byreference), which describes the isolation of high affinity andfunctional agonistic antibodies for MPL- and msk-receptors using such anapproach.

Bispecific or Bifunctional Antigen Binding Proteins

The antigen binding proteins that are provided also include bispecificand bifunctional antibodies that include one or more CDRs or one or morevariable regions as described above. A bispecific or bifunctionalantibody in some instances is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol.79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553.

Various Other Forms

Some of the antigen binding proteins that are provided are variant formsof the antigen binding proteins disclosed above (e.g., those having thesequences listed in TABLES 1-4). For instance, some of the antigenbinding proteins have one or more conservative amino acid substitutionsin one or more of the heavy or light chains, variable regions or CDRslisted in TABLES 1-4.

Naturally-occurring amino acids may be divided into classes based oncommon side chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of the above classes for a member from another class. Suchsubstituted residues may be introduced into regions of the antibody thatare homologous with human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. The hydropathicprofile of a protein is calculated by assigning each amino acid anumerical value (“hydropathy index”) and then repetitively averagingthese values along the peptide chain. Each amino acid has been assigneda hydropathic index on the basis of its hydrophobicity and chargecharacteristics. They are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic profile in conferring interactivebiological function on a protein is understood in the art (see, e.g.,Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In some aspects, those which are within ±1are included, and in other aspects, those within ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigen-binding or immunogenicity, that is, with a biological propertyof the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in otherembodiments, those which are within ±1 are included, and in still otherembodiments, those within ±0.5 are included. In some instances, one mayalso identify epitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary conservative amino acid substitutions are set forth in TABLE5.

TABLE 5 Conservative Amino Acid Substitutions Original Exemplary ResidueSubstitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn GluAsp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu MetLeu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile,Leu

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. Oneskilled in the art may identify suitable areas of the molecule that maybe changed without destroying activity by targeting regions not believedto be important for activity. The skilled artisan also will be able toidentify residues and portions of the molecules that are conserved amongsimilar polypeptides. In further embodiments, even areas that may beimportant for biological activity or for structure may be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues important for activity or structure in similarproteins. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the 3-dimensional structure andamino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. One skilled in the art may choosenot to make radical changes to amino acid residues predicted to be onthe surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. These variants can thenbe screened using assays for c-fms neutralizing activity, (see examplesbelow) thus yielding information regarding which amino acids can bechanged and which must not be changed. In other words, based oninformation gathered from such routine experiments, one skilled in theart can readily determine the amino acid positions where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See, Moult, 1996, Curr. Op. in Biotech.7:422-427; Chou et al., 1974, Biochem. 13:222-245; Chou et al., 1974,Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol. Relat. AreasMol. Biol. 47:45-148; Chou et al., 1979, Ann. Rev. Biochem. 47:251-276;and Chou et al., 1979, Biophys. J. 26:367-384. Moreover, computerprograms are currently available to assist with predicting secondarystructure. One method of predicting secondary structure is based uponhomology modeling. For example, two polypeptides or proteins that have asequence identity of greater than 30%, or similarity greater than 40%can have similar structural topologies. The recent growth of the proteinstructural database (PDB) has provided enhanced predictability ofsecondary structure, including the potential number of folds within apolypeptide's or protein's structure. See, Holm et al., 1999, Nucl.Acid. Res. 27:244-247. It has been suggested (Brenner et al., 1997,Curr. Op. Struct. Biol. 7:369-376) that there are a limited number offolds in a given polypeptide or protein and that once a critical numberof structures have been resolved, structural prediction will becomedramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-387; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskovet al., 1987, Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionarylinkage” (See, Holm, 1999, supra; and Brenner, 1997, supra).

In some embodiments, amino acid substitutions are made that: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterligand or antigen binding affinities, and/or (4) confer or modify otherphysicochemical or functional properties on such polypeptides. Forexample, single or multiple amino acid substitutions (in certainembodiments, conservative amino acid substitutions) may be made in thenaturally-occurring sequence. Substitutions can be made in that portionof the antibody that lies outside the domain(s) forming intermolecularcontacts). In such embodiments, conservative amino acid substitutionscan be used that do not substantially change the structuralcharacteristics of the parent sequence (e.g., one or more replacementamino acids that do not disrupt the secondary structure thatcharacterizes the parent or native antigen binding protein). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed.), 1984, W. H. New York: Freeman and Company; Introduction to ProteinStructure (Branden and Tooze, eds.), 1991, New York: Garland Publishing;and Thornton et al., 1991, Nature 354:105, which are each incorporatedherein by reference.

Additional preferred antibody variants include cysteine variants whereinone or more cysteine residues in the parent or native amino acidsequence are deleted from or substituted with another amino acid (e.g.,serine). Cysteine variants are useful, inter alia when antibodies mustbe refolded into a biologically active conformation. Cysteine variantsmay have fewer cysteine residues than the native antibody, and typicallyhave an even number to minimize interactions resulting from unpairedcysteines.

The heavy and light chains, variable regions domains and CDRs that aredisclosed can be used to prepare polypeptides that contain an antigenbinding region that can specifically bind to a c-fms polypeptide. Forexample, one or more of the CDRs listed in TABLES 3 and 4 can beincorporated into a molecule (e.g., a polypeptide) covalently ornoncovalently to make an immunoadhesion. An immunoadhesion mayincorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDR(s) enable theimmunoadhesion to bind specifically to a particular antigen of interest(e.g., a c-fms polypeptide or epitope thereof).

Mimetics (e.g., “peptide mimetics” or “peptidomimetics”) based upon thevariable region domains and CDRs that are described herein are alsoprovided. These analogs can be peptides, non-peptides or combinations ofpeptide and non-peptide regions. Fauchere, 1986, Adv. Drug Res. 15:29;Veber and Freidinger, 1985, TINS p. 392; and Evans et al., 1987, J. Med.Chem. 30:1229, which are incorporated herein by reference for anypurpose. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce a similartherapeutic or prophylactic effect. Such compounds are often developedwith the aid of computerized molecular modeling. Generally,peptidomimetics are proteins that are structurally similar to anantibody displaying a desired biological activity, such as here theability to specifically bind c-fms, but have one or more peptidelinkages optionally replaced by a linkage selected from: —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH—CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and—CH₂SO—, by methods well known in the art. Systematic substitution ofone or more amino acids of a consensus sequence with a D-amino acid ofthe same type (e.g., D-lysine in place of L-lysine) may be used incertain embodiments to generate more stable proteins. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61:387),incorporated herein by reference), for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

Derivatives of the antigen binding proteins that are described hereinare also provided. The derivatized antigen binding proteins can compriseany molecule or substance that imparts a desired property to theantibody or fragment, such as increased half-life in a particular use.The derivatized antigen binding protein can comprise, for example, adetectable (or labeling) moiety (e.g., a radioactive, colorimetric,antigenic or enzymatic molecule, a detectable bead (such as a magneticor electrodense (e.g., gold) bead), or a molecule that binds to anothermolecule (e.g., biotin or streptavidin)), a therapeutic or diagnosticmoiety (e.g., a radioactive, cytotoxic, or pharmaceutically activemoiety), or a molecule that increases the suitability of the antigenbinding protein for a particular use (e.g., administration to a subject,such as a human subject, or other in vivo or in vitro uses). Examples ofmolecules that can be used to derivatize an antigen binding proteininclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antigen bindingproteins can be prepared using techniques well known in the art. Certainantigen binding proteins include a pegylated single chain polypeptide asdescribed herein. In one embodiment, the antigen binding protein isconjugated or otherwise linked to transthyretin (TTR) or a TTR variant.The TTR or TTR variant can be chemically modified with, for example, achemical selected from the group consisting of dextran, poly(n-vinylpyrrolidone), polyethylene glycols, propropylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyolsand polyvinyl alcohols.

Other derivatives include covalent or aggregative conjugates of c-fmsantigen binding proteins with other proteins or polypeptides, such as byexpression of recombinant fusion proteins comprising heterologouspolypeptides fused to the N-terminus or C-terminus of a c-fms antigenbinding protein. For example, the conjugated peptide may be aheterologous signal (or leader) polypeptide, e.g., the yeastalpha-factor leader, or a peptide such as an epitope tag. C-fins antigenbinding protein-containing fusion proteins can comprise peptides addedto facilitate purification or identification of the c-fms antigenbinding protein (e.g., poly-His). A c-fms antigen binding protein alsocan be linked to the FLAG peptide as described in Hopp et al., 1988,Bio/Technology 6:1204; and U.S. Pat. No. 5,011,912. The FLAG peptide ishighly antigenic and provides an epitope reversibly bound by a specificmonoclonal antibody (mAb), enabling rapid assay and facile purificationof expressed recombinant protein. Reagents useful for preparing fusionproteins in which the FLAG peptide is fused to a given polypeptide arecommercially available (Sigma, St. Louis, Mo.).

Oligomers that contain one or more c-fms antigen binding proteins may beemployed as c-fms antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more c-fms antigen bindingproteins are contemplated for use, with one example being a homodimer.Other oligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiplec-fms-binding polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the c-fms antigen bindingproteins. Such peptides may be peptide linkers (spacers), or peptidesthat have the property of promoting oligomerization. Leucine zippers andcertain polypeptides derived from antibodies are among the peptides thatcan promote oligomerization of c-fms antigen binding proteins attachedthereto, as described in more detail below.

In particular embodiments, the oligomers comprise from two to four c-fmsantigen binding proteins. The c-fms antigen binding protein moieties ofthe oligomer may be in any of the forms described above, e.g., variantsor fragments. Preferably, the oligomers comprise c-fms antigen bindingproteins that have c-fms binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing a c-fms antigen binding protein to the Fc region of anantibody. The dimer can be made by, for example, inserting a gene fusionencoding the fusion protein into an appropriate expression vector,expressing the gene fusion in host cells transformed with therecombinant expression vector, and allowing the expressed fusion proteinto assemble much like antibody molecules, whereupon interchain disulfidebonds form between the Fc moieties to yield the dimer

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151and U.S. Pat. No. 5,426,048 and U.S. Pat. No. 5,262,522, is a singlechain polypeptide extending from the N-terminal hinge region to thenative C-terminus of the Fc region of a human IgG1 antibody. Anotheruseful Fc polypeptide is the Fc mutein described in U.S. Pat. No.5,457,035, and in Baum et al., 1994, EMBO J 13:3992-4001. The amino acidsequence of this mutein is identical to that of the native Fc sequencepresented in WO 93/10151, except that amino acid 19 has been changedfrom Leu to Ala, amino acid 20 has been changed from Leu to Glu, andamino acid 22 has been changed from Gly to Ala. The mutein exhibitsreduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of a c-fms antigen binding protein such as disclosed herein maybe substituted for the variable portion of an antibody heavy and/orlight chain.

Alternatively, the oligomer is a fusion protein comprising multiplec-fms antigen binding proteins, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. No. 4,751,180 and U.S. Pat. No. 4,935,233.

Another method for preparing oligomeric c-fms antigen binding proteinderivatives involves use of a leucine zipper. Leucine zipper domains arepeptides that promote oligomerization of the proteins in which they arefound. Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT applicationWO 94/10308, and the leucine zipper derived from lung surfactant proteinD (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al., 1994, Semin. Immunol. 6:267-278. In oneapproach, recombinant fusion proteins comprising a c-fms antigen bindingprotein fragment or derivative fused to a leucine zipper peptide areexpressed in suitable host cells, and the soluble oligomeric c-fmsantigen binding protein fragments or derivatives that form are recoveredfrom the culture supernatant.

Some antigen binding proteins that are provided have an on-rate (k_(a))for c-fms of at least 10⁴/M× seconds, at least 10⁵/M× seconds, at least10⁶/M× seconds measured, for instance, as described in the examplesbelow. Certain antigen binding proteins that are provided have a slowdissociation rate or off-rate. Some antigen binding proteins, forinstance, have a k_(d) (off-rate) of 1×10⁻² s⁻¹, or 1×10⁻³ s⁻¹, or1×10⁻⁴ s⁻¹, or 1×10⁻⁵ s⁻¹. In certain embodiments, the antigen bindingprotein has a K_(D) (equilibrium binding affinity) of less than 25 pM,50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 25 nM or 50 nM.

Another aspect provides an antigen-binding protein having a half-life ofat least one day in vitro or in vivo (e.g., when administered to a humansubject). In one embodiment, the antigen binding protein has a half-lifeof at least three days. In another embodiment, the antibody or portionthereof has a half-life of four days or longer. In another embodiment,the antibody or portion thereof has a half-life of eight days or longer.In another embodiment, the antibody or antigen-binding portion thereofis derivatized or modified such that it has a longer half-life ascompared to the underivatized or unmodified antibody. In anotherembodiment, the antigen binding protein contains point mutations toincrease serum half life, such as described in WO 00/09560, publishedFeb. 24, 2000, incorporated by reference.

Glycosylation

The antigen-binding protein may have a glycosylation pattern that isdifferent or altered from that found in the native species. As is knownin the art, glycosylation patterns can depend on both the sequence ofthe protein (e.g., the presence or absence of particular glycosylationamino acid residues, discussed below), or the host cell or organism inwhich the protein is produced. Particular expression systems arediscussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence may be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the target polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev, Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Hence, aspects include glycosylation variants of the antigen bindingproteins wherein the number and/or type of glycosylation site(s) hasbeen altered compared to the amino acid sequences of the parentpolypeptide. In certain embodiments, antibody protein variants comprisea greater or a lesser number of N-linked glycosylation sites than thenative antibody. An N-linked glycosylation site is characterized by thesequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X may be any amino acid residue except proline. Thesubstitution of amino acid residues to create this sequence provides apotential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions that eliminate or alter this sequence willprevent addition of an N-linked carbohydrate chain present in the nativepolypeptide. For example, the glycosylation can be reduced by thedeletion of an Asn or by substituting the Asn with a different aminoacid. In other embodiments, one or more new N-linked sites are created.Antibodies typically have a N-linked glycosylation site in the Fcregion.

Labels and Effector Groups

In some embodiments, the antigen-binding comprises one or more labels.The term “labeling group” or “label” means any detectable label.Examples of suitable labeling groups include, but are not limited to,the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g., FITC,rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase),chemiluminescent groups, biotinyl groups, or predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags). In some embodiments, the labeling group iscoupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used as is seen fit.

The term “effector group” means any group coupled to an antigen bindingprotein that acts as a cytotoxic agent. Examples for suitable effectorgroups are radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I). Other suitable groups include toxins,therapeutic group, or chemotherapeutic groups. Examples of suitablegroups include calicheamicin, auristatins, geldanamycin and maytansine.In some embodiments, the effector group is coupled to the antigenbinding protein via spacer arms of various lengths to reduce potentialsteric hindrance.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labeling group iscoupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in MOLECULAR PROBES HANDBOOKby Richard P. Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Labs., Inc., Genbank Accession Number U55762), bluefluorescent protein (BFP, Quantum Biotechnologies, Inc., Quebec, Canada;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Labs.,Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), βgalactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:2603-2607) and Renilla (WO92/15673, WO95/07463, WO98/14605,WO98/26277, WO99/49019, U.S. Pat. No. 5,292,658, U.S. Pat. No.5,418,155, U.S. Pat. No. 5,683,888, U.S. Pat. No. 5,741,668, U.S. Pat.No. 5,777,079, U.S. Pat. No. 5,804,387, U.S. Pat. No. 5,874,304, U.S.Pat. No. 5,876,995, U.S. Pat. No. 5,925,558).

Nucleic Acids Encoding C-fms Antigen Binding Proteins

Nucleic acids that encode for the antigen binding proteins describedherein, or portions thereof, are also provided, including nucleic acidsencoding one or both chains of an antibody, or a fragment, derivative,mutein, or variant thereof, polynucleotides encoding heavy chainvariable regions or only CDRs, polynucleotides sufficient for use ashybridization probes, PCR primers or sequencing primers for identifying,analyzing, mutating or amplifying a polynucleotide encoding apolypeptide, anti-sense nucleic acids for inhibiting expression of apolynucleotide, and complementary sequences of the foregoing. Thenucleic acids can be any length. They can be, for example, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides inlength, and/or can comprise one or more additional sequences, forexample, regulatory sequences, and/or be part of a larger nucleic acid,for example, a vector. The nucleic acids can be single-stranded ordouble-stranded and can comprise RNA and/or DNA nucleotides, andartificial variants thereof (e.g., peptide nucleic acids). TABLE 6 showsexemplary nucleic acid sequences encoding an IgG2 heavy chain constantregion and IgG2 kappa light chain constant region. Any variable regionprovided herein may be attached to these constant regions to formcomplete heavy and light chain sequences. However, it should beunderstood that these constant regions sequences are provided asspecific examples only. In some embodiments, the variable regionsequences are joined to other constant region sequences that are knownin the art. Exemplary nucleic acid sequences encoding heavy and lightchain variable regions are provided in TABLE 7.

TABLE 6Exemplary Nucleic Acid Sequences Encoding Heavy and Light Chain Constant RegionsType Nucleic Acid Sequence/SEQ ID NO. IgG2 heavy chaingctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga [SEQ ID NO: 247] IgG2 kappa lightcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtchaingcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag [SEQ ID NO: 248]

TABLE 7 shows exemplary nucleic acid sequences encoding heavy chain andlight chain variable regions, in which the various CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, and CDRL3 sequences are embedded.

TABLE 7Exemplary Nucleic Acid Sequences Encoding Heavy And Light Chain Variable RegionsReference Designation Nucleic Acid Sequence/SEQ ID NO. H1 109 V_(H)1caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G1cttctggatacaccttcaccgcctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagaggtggatatagtggctacgatttgggctactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 249] H1 13 V_(H)2caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactg1N1G1tctctggtggctccgtcagcagtggtggttactactggagctggatccggcagcccccagggaagggactggagtggattgggtatatctattacagtgggagcaccaactacaacccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttctccctgaagctgagctctgtgaccgctgcggacacggccgtgtattactgtgcggccggtatagcagccactggtaccctctttgactgctggggccagggaaccctggtcaccgtctcctca [SEQ. ID NO: 250] H1 131 V_(H)3caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G1cttctggatacaccttcaccggctactatatacactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagagatcgagggcagctatggttatggtactactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 251] H1 134 V_(H)4caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcag1N1G1cgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgccagcagcagctggtcctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 252] H1 143 V_(H)5gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcag1N1G1cctctggattcactgtcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagacaacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacaggagggtcattactatggaccgggcccaactactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 253] H1 144 V_(H)6gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcag1N1G1cctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagagtactatggttcggggggggtttggtactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 254] H1 16 V_(H)7gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcag1N1G1cctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggttggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagatctccgtataactggaactacctattactactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 255] H1 2 1N1G1 V_(H)8caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggttacacctttaccagctatggtatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcagcgcttacaatggtaacacaaactatgcacagaagctccagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagagagtcgtggttcggggaggtattctttgactactggggccagggaaccctggtcaccgtctcctca [SEQ ID NO: 256] H1 26 V_(H)9gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcag1N1G1cctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagagtactatggttcggggggggtttggtactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 257] H1 27 V_(H)10gaggtgcagctggtgagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcagc1N1G1ctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagatggggctacggtggtaactccggggtactactactacggtacggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 258] H1 30 V_(H)11caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G1cttctggttacacctttaccagctatggtatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcagcgcttacaatggtaacacaaactatgcacagaagctccagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagagagtcgtggttcggggaggtattttttgactactggggccagggaaccctggtcaccgtctcctca [SEQ ID NO: 259] H1 33 1- V_(H)12caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G1cttctggatacaccttcaccggctactatatgacactgggtgcgacaggcccctggacaagggcttgaatggatgggatggatcaaccctaacagtggtggcacaaactatgctcagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcagactgagatctgacgacacggccttttattactgtgcgagagacagcaactggtaccacaactggttcgacccctggggccagggaaccctggtcaccgtctcctca [SEQ ID NO: 260] H1 33 V_(H)13caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G1cttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgaatggatgggatggatcaaccctaacagtggtggcacaaactatgctcagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcagactgagatctgacgacacggccttttattactgtgcgagagacagcaactggtaccacaactggttcgacccctggggccagggaaccctggtcaccgtctcctca [SEQ ID NO: 261] H1 34 V_(H)14gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcag1N1G1cctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagatggggctacggtggtaactccggggtactactactacggtacggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 262] H1 39 V_(H)15gaggtgcaactggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcag1N1G1cctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacagcagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagaaggtccctacagtgactacgggtactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 263] H1 42 V_(H)16caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G1cttctggttacacctttaccagctatggtatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcagcgcttacaatggtaacacaaactatgcacagaagctccagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagagagtcgtggttcggggaggtattctttgactactggggccagggaaccctggtcaccgtctcctca [SEQ ID NO: 264] H1 64 V_(H)17gaggtgcagctggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcag1N1G1cctctggattcaccttcagtagctacgacatgcactgggtccgccaagctacaggaaaaggtctggagtgggtctcaggtattggtactgctggtgacacatactatccaggctccgtgaagggccgattcaacatctccagagaaaatgccaagaactccttgtatcttcaaatgaacagcctgagagccggggacacggctgtgtattactgtgcaagagagggcagctggtacggctttgactactggggccagggaaccctggtcaccgtctcctc a[SEQ ID NO: 265] H1 66 V_(H)18caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcag1N1G1cgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaatgaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagagcacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgcactcgtccgggaactactacgatatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 266] V_(H)19 V_(H)19gaggtgcagctggtggagtctgggggaggcttggtagagcctggggggtcccttagactctcctgtgcagcctctggattcactttcagtaccgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaacgaggacacagccgtgtattactgtaccacagaaggtccctacagtaactacgggtactactactacggtgtggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 267] H1 90 V_(H)20Caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcag1N1G1cgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagcagctcgtcaaacttctacgatatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 268] H2 103 V_(H)21gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttacactctcctgtgcag1N1G2cctctggattcactttcaataacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggttggccgtattaaaagcaaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagaatattaccatattttgactggttcgttctactactcctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 269] H2 131 V_(H)22caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactg1N1G2tctctggtggctccatcagtaattactactggagctggatccggcagtccgccgggaagggactggagtggattgggcgtatctataccagtgggagcacccactacaacccctccctcaagagtcgaatcatcatgtcagtggacacgtccaagaaccagttctccctgaagctgagctctgtgaccgccgcggacacggccgtgtattactgtgcgagagatcgagtcttctacggtatggacgtctggggccaagggaccacggtcaccgtctcctc a[SEQ ID NO: 270] H2 291 V_(H)23caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcag1N1G2cgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagttataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagaaggggattactccgactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 271] H2 360 V_(H)24caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G2tttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagtttacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaacaggggttatgattacgtttgggggagttatcgttggccactcctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 272] H2 369 V_(H)26caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G2tttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaacaagggctggaacgacgttggcctactactactacgctatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 273] H2 380 V_(H)27caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactg1N1G2tctctggtggctccatcagtagttactactggagctggatccggcagcccccagggaagggactggagtggattgggtatatctattacagtgggaacaccaactacaacccctccctcaagagtcgattcaccttatcaatagacacgtccaagaaccagttctccctgaggctgagctctgtgaccgctgcggacacggccgtgtattactgtgcgtgtatagcaactcggccctttgactactggggccagggaaccctggtcaccgtctcctca[SEQ ID NO: 274] H2 475 V_(H)28caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcag1N1G2cgtcaggattcaccttcatcagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcggatagcagtggcgactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 275] H2 508 V_(H)29caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G2tttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctatatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaacagcggggctggaaatacggtggttcgacccctggggccagggaaccctggtcaccgtctcctca [SEQ ID NO: 276] H2 534 V_(H)30caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactg1N1G2tctctggtggctccatcagcagtggtggttactactggagctggatccgccagcacccagggaagggcctggagtggattgggtacatctcttacagtggggacacctactacaacccgtccctcaagagtcgacttaccatatcagtagacacgtctaagcaccagttctccctgaggctgagctctgtgacttccgcggacacggccgtgtattactgtgcgagtctagacctctacggtgactactttgactactggggccagggaaccctggtcaccgtctcctca [SEQ ID NO: 277] H2 550 V_(H)31caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaagg1N1G2cttctggttacaccttaaccagctatggtatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcagcgcttacaatggtaacccaaactatgcacagaagttccagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagagatcagggattactagggttcggggaactcgaggggctctttgactactggggccagggaaccctggtcaccgtctcctca [SEQ ID NO: 278] H2 65 V_(H)32gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgcag1N1G2cctctggattcactttcagtaacgcctggatgagctgggtccgccaggctccagggaaggggctggagtgggtggccgtattaaaaccaaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcacaaaacacgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattactgtaccacagaatattacggtattgtgactggttcgttttattactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca [SEQ ID NO: 279] H1 109 V_(L)1gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaaaacattagcaactttttagattggtatcagcagaaaccagggaaagcccctaacctcctgatctacgatgcatccgatttggatccaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctacagcctgaagatattgcaacatattactgtcaacagtatgttagtctcccgctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 280] H1 131 V_(L)3gataatgtgatgacccagactccactctctctgtccgtcacccctggacagccggcctccatctcctgca1N1Kagtcgagtcagagcctcctgcatagtgatgggaagacctatttgtattggtacctgcagaagccaggccagcctccacagctcctgatctatgaagcttccaaccggttctctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatcagccgggtggaggctgaggatgttggggtttattactgcatgcaaagtatacagcttcctctcactttcggcggagggaccaaggtggagatcaaa[SEQ ID NO: 281] H1 134 V_(L)4gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattaacaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctacgatgcatccaatttggaaataggggtcccatcaaggttcagtggaatggatctgggacagatttcattttcaccatcagcagtctgcagcctgaagatattgcaacatattactgtcaacagtatgataatttcccgttcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 282] H1 143 V_(L)5gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattaacaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctacgatacatccaatttggaaccaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacaatatgataatctcctcaccttcggccaagggacacgactggaaattaaa [SEQ ID NO: 283] H1 144 V_(L)6gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactatttaaattggtatcagcataaaccagggaaagcccctaaactcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacagtatgataatctgctcaccttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 284] H1 16 V_(L)7gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagttcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggtttagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacagtatgataatctgatcaccttcggccaagggacacgactggagattaaa [SEQ ID NO: 285] H1 2 V_(L)8gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgca1N1Kagtccagccagagtgttttagacagctccgacaataagaactacttagcttggtaccagcagaaaccaggacagcctcctaagctgctcatttactgggcatctaaccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttctctctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaatattatagtgatccattcactttcggccctgggaccaaagtggatatcaaa[SEQ ID NO: 286] H1 27 V_(L)10gacatccagatgacccagtctccatcctccctgtctgcatctgtaggtgacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactatttaaattggtatcaacagaaaccagggaaagcccctaaactcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctacagcctgaagatattgcaacatattactgtcaacagtatgataatctgctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 287] H1 30 V_(L)11gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcgactgca1N1Kagtccagccagggtgttttagacagctccaacaataagaacttcttagcttggtaccagcagaaaccaggacagcctccaagctgctcatttactgggcatctaaccgggaatccggggtccctgtccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcactttattactgtcagcaatattatagtgatccattcactttcggccctgggaccaaagtggatatcaaa[SEQ ID NO: 288] H1 33-1 V_(L)12gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kgggcaagtcagagcattagtgactatttaaattggtatcagcagaaaccagggaaagcccctaacctcctgatctatgctgcatccagtttgcagagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttacttctgtcaacagacttacagtgacccattcactttcggccctgggaccaaagtggatatcaaa [SEQ ID NO: 289] H1 34 V_(L)13gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctacagcctgaagatattgcaacatattactgtcaacagtatgataatctgctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 290] H1 39 V_(L)14gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaaggtcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacagtatgataatctcctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 291] H1 42 V_(L)15gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcgactgca1N1Kagtccagccagagtgttttagacagctccaacaataagaacttcttagcttggtaccagcagaaaccaggacagcctcctaagctgctcatttactgggcatctaaccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaatattatagtgatccattcactttcggccctgggaccaaagtggatatcaaa[SEQ ID NO: 292] H1 64 V_(L)16gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgca1N1Kgggccagtcagagtgttagcagcggctacttagcctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcacggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtagctcaccgatcaccttcggccaagggacacgactggagattaaa [SEQ ID NO: 293]H1 66 V_(L)17gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactttttaaattggtatcagcagagaccagggaaagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacagtatgataatctcccattcactttcggccctgggaccaaagtggatatcaaa [SEQ ID NO: 294] H1 72 V_(L)18gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaaactcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagattttgcaacatattactgtcaacagtatgataatctcctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 295] H1 90 V_(L)19gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccaggaaaagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacggtatgatgatctcccgatcaccttcggccaagggacacgactggagattaaa [SEQ ID NO: 296] H2 103V_(L)20gacatccagatgacccagtctccatcctccctgtctgcatctgtgggagacagagtcaccatcacttgcc1N1Kaggcgagtcaggacattagcaactatttaaattggtatcagcagagaccagggaaagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacagtatgataatctgctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 297] H2 131 V_(L)21gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kgggcgagtcagggctttagcaattatttagcctggtatcagcagaaaccagggaaagttcctaagctcctgatctatgctgcatccactttgcagtcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaagtataacagtgccccgctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 298] H2 360V_(L)22gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kgggcgagtcagggcattaacaattatttagcctggtatcagcagaaaccagggaaagttcctcagctcctgatctatgttgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaagtataacagtggcccattcactttcggccctgggaccaaagtggatatcaaa [SEQ ID NO: 299] H2 369V_(L)24gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kgggcaagtcagagcattagcaggtatttaaattggtatcagcagaaaccagggaaagcccctaacctcctgatccatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacattacccctcccagttttggccaggggaccaagctggagatcaaa [SEQ ID NO: 300] H2 380V_(L)25gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1Kgggcaagtcagggcattagaaatgatttagactggtatcagcagaaaccagggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatctaggttcagcggcagtggatctgggacagaattcactctcacaatcaacagcctgcagcctgaagattttgcaacttattactgtctacagtataatagttacccgatcaccttcggccaagggacacgactggagattaaa [SEQ ID NO: 301] H2 475V_(L)26gacatccagatgatccagtctccttcctccctgtctgcatctgtcggagacagagtcaccatcacttgcc1N1Kaggcgagtcacgacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagttcctgatctccgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacagtatgataatctcccgctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 302] H2 508 H2 508gatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgca1N1K 1N1KggtctagtcagagcctcctgcatagtaatggatacaactatttggattggtacctgcagaagccagggcaV_(L)27 V_(L)27gtcaccacagttcctgatctatttgggttctattcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttgcactgacaatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactcctcggacgttcggccaagggaccaaggtggaaatcaaa[SEQ ID NO: 303] H2 534 H2 534gaaattgtgctgactcagtctccagactttcagtctgtgactccaaaggagaaagtcaccatcacctgcc1N1K 1N1KgggccagtcagatacattggtagtagcttacactggtaccagcagacaccagatcagtctccaaagctccV_(L)28 V_(L)28tcatcaactatgtttcccagtccttctcaggggtcccctcgaggttcagtggcagtggatctgggacagatttcaccctcaccatcaatagcctggaagctgaagatgctgcaacgtattactgtcatcagagtagtagtttaccattcactttcggccctgggaccaaagtggatatcaaa [SEQ ID NO: 304] H2 550H2 550gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgcgagggccaccatctcctgca1N1K 1N1KagtccagccagagtgttttatacagctccaacaataagaactacttagcttggtaccagcagaaaccaggV_(L)29 V_(L)29ccagcctcctaagctgctcatttactgggcatctacccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcaccctgcaggctgaagatgtggcagtttattactgtcagcaatattatactactcctccgacgttcggccaagggaccaaggtggaaatcaaa[SEQ ID NO: 305] H2 65 H2 65gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcc1N1K 1N1KaggcgagtcaggacattaacaactatttaaattggtatcaacagaaaccagggaaagcccctaaactcctV_(L)30 V_(L)30gatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacagtatgatgatctgctcactttcggcggagggaccaaggtggagatcaaa [SEQ ID NO: 306]

Nucleic acids encoding certain antigen binding proteins, or portionsthereof (e.g., full length antibody, heavy or light chain, variabledomain, or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) may be isolatedfrom B-cells of mice that have been immunized with c-fms or animmunogenic fragment thereof. The nucleic acid may be isolated byconventional procedures such as polymerase chain reaction (PCR). Phagedisplay is another example of a known technique whereby derivatives ofantibodies and other antigen binding proteins may be prepared. In oneapproach, polypeptides that are components of an antigen binding proteinof interest are expressed in any suitable recombinant expression system,and the expressed polypeptides are allowed to assemble to form antigenbinding protein molecules.

The nucleic acids provided in TABLES 6 and 7 are exemplary only. Due tothe degeneracy of the genetic code, each of the polypeptide sequenceslisted in TABLES 1-4 or otherwise depicted herein are also encoded by alarge number of other nucleic acid sequences besides those provided. Oneof ordinary skill in the art will appreciate that the presentapplication thus provides adequate written description and enablementfor each degenerate nucleotide sequence encoding each antigen bindingprotein.

An aspect further provides nucleic acids that hybridize to other nucleicacids (e.g., nucleic acids comprising a nucleotide sequence listed inTABLE 6 and TABLE 7) under particular hybridization conditions. Methodsfor hybridizing nucleic acids are well-known in the art. See, e.g.,Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. As defined herein, a moderately stringent hybridizationcondition uses a prewashing solution containing 5× sodiumchloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6×SSC, and a hybridizationtemperature of 55° C. (or other similar hybridization solutions, such asone containing about 50% formamide, with a hybridization temperature of42° C.), and washing conditions of 60° C., in 0.5×SSC, 0.1% SDS. Astringent hybridization condition hybridizes in 6×SSC at 45° C.,followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C.Furthermore, one of skill in the art can manipulate the hybridizationand/or washing conditions to increase or decrease the stringency ofhybridization such that nucleic acids comprising nucleotide sequencesthat are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%identical to each other typically remain hybridized to each other.

The basic parameters affecting the choice of hybridization conditionsand guidance for devising suitable conditions are set forth by, forexample, Sambrook, Fritsch, and Maniatis (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., supra; and Current Protocols in Molecular Biology, 1995,Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4), and can be readily determined by those having ordinary skillin the art based on, e.g., the length and/or base composition of thenucleic acid.

Changes can be introduced by mutation into a nucleic acid, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anantibody or antibody derivative) that it encodes. Mutations can beintroduced using any technique known in the art. In one embodiment, oneor more particular amino acid residues are changed using, for example, asite-directed mutagenesis protocol. In another embodiment, one or morerandomly selected residues is changed using, for example, a randommutagenesis protocol. However it is made, a mutant polypeptide can beexpressed and screened for a desired property.

Mutations can be introduced into a nucleic acid without significantlyaltering the biological activity of a polypeptide that it encodes. Forexample, one can make nucleotide substitutions leading to amino acidsubstitutions at non-essential amino acid residues. Alternatively, oneor more mutations can be introduced into a nucleic acid that selectivelychanges the biological activity of a polypeptide that it encodes. Forexample, the mutation can quantitatively or qualitatively change thebiological activity. Examples of quantitative changes includeincreasing, reducing or eliminating the activity. Examples ofqualitative changes include changing the antigen specificity of anantibody. In one embodiment, a nucleic acid encoding any antigen bindingprotein described herein can be mutated to alter the amino acid sequenceusing molecular biology techniques that are well-established in the art.Example 4, for instance, describes how nucleic acid sequences (see Table6) were mutated to introduce one or more amino acid substitutions intocertain antigen binding proteins to produce antigen binding proteins 1.2SM 1.109 SM and 2.360 SM. Additional antigen binding proteins containingother mutations can be produced in a similar way.

Another aspect provides nucleic acid molecules that are suitable for useas primers or hybridization probes for the detection of nucleic acidsequences. A nucleic acid molecule can comprise only a portion of anucleic acid sequence encoding a full-length polypeptide, for example, afragment that can be used as a probe or primer or a fragment encoding anactive portion (e.g., a c-fms binding portion) of a polypeptide.

Probes based on the sequence of a nucleic acid can be used to detect thenucleic acid or similar nucleic acids, for example, transcripts encodinga polypeptide. The probe can comprise a label group, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used to identify a cell that expresses thepolypeptide.

Another aspect provides vectors comprising a nucleic acid encoding apolypeptide or a portion thereof (e.g., a fragment containing one ormore CDRs or one or more variable region domains). Examples of vectorsinclude, but are not limited to, plasmids, viral vectors, non-episomalmammalian vectors and expression vectors, for example, recombinantexpression vectors. The recombinant expression vectors can comprise anucleic acid in a form suitable for expression of the nucleic acid in ahost cell. The recombinant expression vectors include one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operably linked to the nucleic acid sequence tobe expressed. Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter andcytomegalovirus promoter), those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences, see, Voss et al., 1986, Trends Biochem. Sci.11:287, Maniatis et al., 1987, Science 236:1237, incorporated byreference herein in their entireties), and those that direct inducibleexpression of a nucleotide sequence in response to particular treatmentor condition (e.g., the metallothionin promoter in mammalian cells andthe tet-responsive and/or streptomycin responsive promoter in bothprokaryotic and eukaryotic systems (see, id.). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. The expression vectorscan be introduced into host cells to thereby produce proteins orpeptides, including fusion proteins or peptides, encoded by nucleicacids as described herein.

Another aspect provides host cells into which a recombinant expressionvector has been introduced. A host cell can be any prokaryotic cell (forexample, E. coli) or eukaryotic cell (for example, yeast, insect, ormammalian cells (e.g., CHO cells)). Vector DNA can be introduced intoprokaryotic or eukaryotic cells via conventional transformation ortransfection techniques. For stable transfection of mammalian cells, itis known that, depending upon the expression vector and transfectiontechnique used, only a small fraction of cells may integrate the foreignDNA into their genome. In order to identify and select these integrants,a gene that encodes a selectable marker (e.g., for resistance toantibiotics) is generally introduced into the host cells along with thegene of interest. Preferred selectable markers include those whichconfer resistance to drugs, such as G418, hygromycin and methotrexate.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die), amongother methods.

Preparing of Antigen Binding Proteins

Non-human antibodies that are provided can be, for example, derived fromany antibody-producing animal, such as mouse, rat, rabbit, goat, donkey,or non-human primate (such as monkey (e.g., cynomologous or rhesusmonkey) or ape (e.g., chimpanzee)). Non-human antibodies can be used,for instance, in in vitro cell culture and cell-culture basedapplications, or any other application where an immune response to theantibody does not occur or is insignificant, can be prevented, is not aconcern, or is desired. In certain embodiments, the antibodies may beproduced by immunizing with full-length c-fms or with the extracellulardomain of c-fms. Alternatively, the certain non-human antibodies may beraised by immunizing with amino acids which are segments of c-fms thatform part of the epitope to which certain antibodies provided hereinbind (see infra). The antibodies may be polyclonal, monoclonal, or maybe synthesized in host cells by expressing recombinant DNA.

Fully human antibodies may be prepared as described above by immunizingtransgenic animals containing human immunoglobulin loci or by selectinga phage display library that is expressing a repertoire of humanantibodies.

The monoclonal antibodies (mAbs) can be produced by a variety oftechniques, including conventional monoclonal antibody methodology,e.g., the standard somatic cell hybridization technique of Kohler andMilstein, 1975, Nature 256:495. Alternatively, other techniques forproducing monoclonal antibodies can be employed, for example, the viralor oncogenic transformation of B-lymphocytes. One suitable animal systemfor preparing hybridomas is the murine system, which is a very wellestablished procedure. Immunization protocols and techniques forisolation of immunized splenocytes for fusion are known in the art. Forsuch procedures, B cells from immunized mice are fused with a suitableimmortalized fusion partner, such as a murine myeloma cell line. Ifdesired, rats or other mammals besides can be immunized instead of miceand B cells from such animals can be fused with the murine myeloma cellline to form hybridomas. Alternatively, a myeloma cell line from asource other than mouse may be used. Fusion procedures for makinghybridomas also are well known.

The single chain antibodies that are provided may be formed by linkingheavy and light chain variable domain (Fv region) fragments via an aminoacid bridge (short peptide linker), resulting in a single polypeptidechain. Such single-chain Fvs (scFvs) may be prepared by fusing DNAencoding a peptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85:5879; Ward et al., 1989, Nature 334:544, de Graafet al., 2002, Methods Mol. Biol. 178:379-387. Single chain antibodiesderived from antibodies provided herein include, but are not limited toscFvs comprising the variable domain combinations of the heavy and lightchain variable regions depicted in TABLE 2, or combinations of light andheavy chain variable domains which include CDRs depicted in TABLES 3 and4.

Antibodies provided herein that are of one subclass can be changed toantibodies from a different subclass using subclass switching methods.Thus, IgG antibodies may be derived from an IgM antibody, for example,and vice versa. Such techniques allow the preparation of new antibodiesthat possess the antigen binding properties of a given antibody (theparent antibody), but also exhibit biological properties associated withan antibody isotype or subclass different from that of the parentantibody. Recombinant DNA techniques may be employed. Cloned DNAencoding particular antibody polypeptides may be employed in suchprocedures, e.g., DNA encoding the constant domain of an antibody of thedesired isotype. See, e.g., Lantto et al., 2002, Methods Mol. Biol.178:303-316.

Accordingly, the antibodies that are provided include those comprising,for example, the variable domain combinations described, supra., havinga desired isotype (for example, IgA, IgG1, IgG2, IgG3, IgG4, IgE, andIgD) as well as Fab or F(ab′)₂ fragments thereof. Moreover, if an IgG4is desired, it may also be desired to introduce a point mutation(CPSCP→CPPCP) in the hinge region as described in Bloom et al., 1997,Protein Science 6:407, incorporated by reference herein) to alleviate atendency to form intra-H chain disulfide bonds that can lead toheterogeneity in the IgG4 antibodies.

Moreover, techniques for deriving antibodies having different properties(i.e., varying affinities for the antigen to which they bind) are alsoknown. One such technique, referred to as chain shuffling, involvesdisplaying immunoglobulin variable domain gene repertoires on thesurface of filamentous bacteriophage, often referred to as phagedisplay. Chain shuffling has been used to prepare high affinityantibodies to the hapten 2-phenyloxazol-5-one, as described by Marks etal., 1992, BioTechnology 10:779.

Conservative modifications may be made to the heavy and light chainvariable regions described in TABLE 2, or the CDRs described in TABLE 3and 4 (and corresponding modifications to the encoding nucleic acids) toproduce a c-fms antigen binding protein having functional andbiochemical characteristics. Methods for achieving such modificationsare described above.

C-fms antigen binding proteins may be further modified in various ways.For example, if they are to be used for therapeutic purposes, they maybe conjugated with polyethylene glycol (pegylated) to prolong the serumhalf-life or to enhance protein delivery. Alternatively, the V region ofthe subject antibodies or fragments thereof may be fused with the Fcregion of a different antibody molecule. The Fc region used for thispurpose may be modified so that it does not bind complement, thusreducing the likelihood of inducing cell lysis in the patient when thefusion protein is used as a therapeutic agent. In addition, the subjectantibodies or functional fragments thereof may be conjugated with humanserum albumin to enhance the serum half-life of the antibody or fragmentthereof. Another useful fusion partner for the antigen binding proteinsor fragments thereof is transthyretin (TTR). TTR has the capacity toform a tetramer, thus an antibody-TTR fusion protein can form amultivalent antibody which may increase its binding avidity.

Alternatively, substantial modifications in the functional and/orbiochemical characteristics of the antigen binding proteins describedherein may be achieved by creating substitutions in the amino acidsequence of the heavy and light chains that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulkiness of the side chain. A “conservativeamino acid substitution” may involve a substitution of a native aminoacid residue with a normative residue that has little or no effect onthe polarity or charge of the amino acid residue at that position. See,TABLE 3, supra. Furthermore, any native residue in the polypeptide mayalso be substituted with alanine, as has been previously described foralanine scanning mutagenesis.

Amino acid substitutions (whether conservative or non-conservative) ofthe subject antibodies can be implemented by those skilled in the art byapplying routine techniques. Amino acid substitutions can be used toidentify important residues of the antibodies provided herein, or toincrease or decrease the affinity of these antibodies for human c-fms orfor modifying the binding affinity of other antigen-binding proteinsdescribed herein.

Methods of Expressing Antigen Binding Proteins

Expression systems and constructs in the form of plasmids, expressionvectors, transcription or expression cassettes that comprise at leastone polynucleotide as described above are also provided herein, as wellhost cells comprising such expression systems or constructs.

The antigen binding proteins provided herein may be prepared by any of anumber of conventional techniques. For example, c-fms antigen bindingproteins may be produced by recombinant expression systems, using anytechnique known in the art. See, e.g., Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.)Plenum Press, New York (1980); and Antibodies: A Laboratory Manual,Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988).

Antigen binding proteins can be expressed in hybridoma cell lines (e.g.,in particular antibodies may be expressed in hybridomas) or in celllines other than hybridomas. Expression constructs encoding theantibodies can be used to transform a mammalian, insect or microbialhost cell. Transformation can be performed using any known method forintroducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus or bacteriophage and transducinga host cell with the construct by transfection procedures known in theart, as exemplified by U.S. Pat. No. 4,399,216; U.S. Pat. No. 4,912,040;U.S. Pat. No. 4,740,461; U.S. Pat. No. 4,959,455. The optimaltransformation procedure used will depend upon which type of host cellis being transformed. Methods for introduction of heterologouspolynucleotides into mammalian cells are well known in the art andinclude, but are not limited to, dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, mixing nucleic acid with positively-charged lipids, anddirect microinjection of the DNA into nuclei.

Recombinant expression constructs typically comprise a nucleic acidmolecule encoding a polypeptide comprising one or more of the following:one or more CDRs provided herein; a light chain constant region; a lightchain variable region; a heavy chain constant region (e.g., C_(H)1,C_(H)2 and/or C_(H)3); and/or another scaffold portion of a c-fmsantigen binding protein. These nucleic acid sequences are inserted intoan appropriate expression vector using standard ligation techniques. Inone embodiment, the heavy or light chain constant region is appended tothe C-terminus of the anti-c-fms-specific heavy or light chain variableregion and is ligated into an expression vector. The vector is typicallyselected to be functional in the particular host cell employed (i.e.,the vector is compatible with the host cell machinery, permittingamplification and/or expression of the gene can occur). In someembodiments, vectors are used that employ protein-fragmentcomplementation assays using protein reporters, such as dihydrofolatereductase (see, for example, U.S. Pat. No. 6,270,964, which is herebyincorporated by reference). Suitable expression vectors can bepurchased, for example, from Invitrogen Life Technologies or BDBiosciences (formerly “Clontech”). Other useful vectors for cloning andexpressing the antibodies and fragments include those described inBianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. 84:439-44, whichis hereby incorporated by reference. Additional suitable expressionvectors are discussed, for example, in Methods Enzymol., vol. 185 (D. V.Goeddel, ed.), 1990, New York: Academic Press.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the c-fmsantigen binding protein coding sequence; the oligonucleotide sequenceencodes polyHis (such as hexaHis), or another “tag” such as FLAG®, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the c-fms antigen binding protein from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified c-fms antigen binding protein by various means such as usingcertain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors may be obtained by any ofseveral methods well known in the art. Typically, flanking sequencesuseful herein will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein that binds to c-fms polypeptide. As a result,increased quantities of a polypeptide such as an antigen binding proteinare synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orpro-sequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein), one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning will typically contain a promoter that isrecognized by the host organism and operably linked to the moleculeencoding c-fms antigen binding protein. Promoters are untranscribedsequences located upstream (i.e., 5′) to the start codon of a structuralgene (generally within about 100 to 1000 bp) that control transcriptionof the structural gene. Promoters are conventionally grouped into one oftwo classes: inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature.Constitutive promoters, on the other hand, uniformly transcribe a geneto which they are operably linked, that is, with little or no controlover gene expression. A large number of promoters, recognized by avariety of potential host cells, are well known. A suitable promoter isoperably linked to the DNA encoding heavy chain or light chaincomprising a c-fms antigen binding protein by removing the promoter fromthe source DNA by restriction enzyme digestion and inserting the desiredpromoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus, and Simian Virus 40(SV40). Other suitable mammalian promoters include heterologousmammalian promoters, for example, heat-shock promoters and the actinpromoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene (Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising ahuman c-fms antigen binding protein by higher eukaryotes. Enhancers arecis-acting elements of DNA, usually about 10-300 bp in length, that acton the promoter to increase transcription. Enhancers are relativelyorientation and position independent, having been found at positionsboth 5′ and 3′ to the transcription unit. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Typically, however, anenhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer may be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter. A sequence encoding an appropriate native orheterologous signal sequence (leader sequence or signal peptide) can beincorporated into an expression vector, to promote extracellularsecretion of the antibody. The choice of signal peptide or leaderdepends on the type of host cells in which the antibody is to beproduced, and a heterologous signal sequence can replace the nativesignal sequence. Examples of signal peptides that are functional inmammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al. 1984,Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The expression vectors that are provided may be constructed from astarting vector such as a commercially available vector. Such vectorsmay or may not contain all of the desired flanking sequences. Where oneor more of the flanking sequences described herein are not alreadypresent in the vector, they may be individually obtained and ligatedinto the vector. Methods used for obtaining each of the flankingsequences are well known to one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising a c-fms antigen binding sequence has been inserted into theproper site of the vector, the completed vector may be inserted into asuitable host cell for amplification and/or polypeptide expression. Thetransformation of an expression vector for an antigen-binding proteininto a selected host cell may be accomplished by well known methodsincluding transfection, infection, calcium phosphate co-precipitation,electroporation, microinjection, lipofection, DEAE-dextran mediatedtransfection, or other known techniques. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., 2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes anantigen binding protein that can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines may be selected throughdetermining which cell lines have high expression levels andconstitutively produce antigen binding proteins with c-fms bindingproperties. In another embodiment, a cell line from the B cell lineagethat does not make its own antibody but has a capacity to make andsecrete a heterologous antibody can be selected.

Use of Human C-fms Antigen Binding Proteins for Diagnostic andTherapeutic Purposes

Antigen binding proteins are useful for detecting c-fms in biologicalsamples and identification of cells or tissues that produce c-fms. Forinstance, the c-fms antigen binding proteins can be used in diagnosticassays, e.g., binding assays to detect and/or quantify c-fms expressedin a tissue or cell. Antigen binding proteins that specifically bind toc-fms can also be used in treatment of diseases related to c-fms in apatient in need thereof. In addition, c-fms antigen binding proteins canbe used to inhibit c-fms from forming a complex with its ligand CSF-1,thereby modulating the biological activity of c-fms in a cell or tissue.Examples of activities that can be modulated include, but are notlimited to, inhibiting autophosphorylation of c-fms, reducing monocytechemotaxis, inhibiting monocyte migration, inhibiting the accumulationof tumor associated macrophages in a tumor or diseased tissue and/orinhibiting angiogenesis. Antigen binding proteins that bind to c-fmsthus can modulate and/or block interaction with other binding compoundsand as such may have therapeutic use in ameliorating diseases related toc-fms.

Indications

Many tumor cells secrete CSF-1 that, in turn, attracts, promotes thesurvival of, and activates monocyte/macrophage cells through the cognatereceptor c-fms. The level of CSF-1 in human tumors has been shown tocorrelate positively with the number of TAMs present in those tumors(Murdoch et al., 2004, Blood 104:2224-2234). Several studies have linkedhigh TAM numbers with reduced patient survival in patients with variousforms of cancer. Recent studies have indicated the existence of anautocrine loop in tumor cells. Other research indicates that c-fms playsa role in various inflammatory diseases. Therefore, regulation ofc-fms-CSF-1 signaling by the human c-fms antigen-binding proteinsprovided herein can inhibit, interfere with, or modulate at least one ofthe biological responses related to c-fms, and, as such, are useful forameliorating the effects of c-fms-related diseases or conditions. C-fmsbinding proteins provided herein can also be used for the diagnosis,prevention or treatment of such diseases or conditions.

A disease or condition associated with human c-fms includes any diseaseor condition whose onset in a patient is caused by, at least in part,the interaction of c-fms with the CSF-1 ligand and/or IL-34. Theseverity of the disease or condition can also be increased or decreasedby the interaction of c-fms with CSF-1 and/or IL-34. Examples ofdiseases and conditions that can be treated with the antigen bindingproteins include various cancers, inflammatory diseases and bonedisorders. The antigen binding proteins can also be used to treat orprevent metastasis of cancer and bone osteolysis associated with themetastasis of cancer to bone.

A high level of TAMs is associated with tumor growth in a variety ofcancers, including: breast (Tsutsui et al., 2005, Oncol. Rep.14:425-431; Leek et al., 1999, Br. J. Cancer 79:991-995; Leek andHarris, 2002, J. Mammary Gland Biol and Neoplasia 7:177-189), prostate(Lissbrant et al. 2000, Int. J. Oncol. 17:445-451), endometrial (Ohno etal., 2004, Anticancer Res. 24:3335-3342), bladder (Hanada et al., 2000,Int. J. Urol 7:263-269), kidney (Hamada et al.; 2002, Anticancer Res.22:4281-4284), esophageal (Lewis and Pollard, 2006, Cancer Res.66(2):606-612), squamous cell (Koide et al., 2004, Am. J. Gastroenterol.99:1667-1674), uveal melanoma (Makitie et al., 2001, Invest. Ophthalmol.Vis. Sci. 42:1414-1421), follicular lymphoma (Farinha et al., 2005,Blood 106:2169-2174), renal and cervical (Kirma et al., 2007, Cancer Res67: 1918-1926). In the cases of breast cancer, prostate cancer,endometrial cancer, bladder cancer, kidney cancer, esophageal cancer,squamous cell carcinoma, uveal melanoma, follicular lymphomas andovarian cancer, the high levels of TAMs also indicates reduced patientsurvival. Therefore, the c-fms antigen binding proteins provided hereincan be used to inhibit recruitment to and decrease survival and functionof the TAMs in the tumor, thus negatively affecting tumor growth, andincreasing patient survival.

Other cancers that can be treated include, but are not limited to, solidtumors generally, lung cancer, ovarian cancer, colorectal cancer, brain,pancreatic, head, neck, liver, leukemia, lymphoma and Hodgkin's disease,multiple myeloma (Farinha et al., 2005, Blood 106:2169-2174), melanoma,gastric cancer, astrocytic cancer, stomach, and pulmonaryadenocarcinoma. Ishigami et al., 2003, Anticancer Research 23:4079-4083;Caruso et al., 1999, Modern Pathology 12:386-390; Witcher et al., 2004,Research Support 104:3335-3342; Haran-Ghera et al. 1997, Blood89:2537-2545; Hussein et al., 2006 International Journal of ExperimentalPathology 87:163-76; Lau et al. 2006 British Journal of Cancer.94:1496-1503, Leung et al. 1997, Acta Neuropathologica. 93:518-527,Giraudo et al., 2004, Journal of Clinical Investigation 114:623-633;Kirma et al., 2007, Cancer Research 67:1918-26, van Ravenswaay et al.,1992, Laboratory Investigation 67:166-174.

The antigen binding proteins can also be used to inhibit tumor growth,progression and/or metastasis. Such inhibition can be monitored usingvarious methods. For instance, inhibition can result in reduced tumorsize and/or a decrease in metabolic activity within the tumor. Both ofthese parameters can be measured by MRI or PET scans for example.Inhibition can also be monitored by biopsy to ascertain the level ofnecrosis, tumor cell death and the level of vascularity within thetumor. The extent of metastasis and bone osteolysis associated withmetastasis can be monitored using known methods.

Evidence for the existence of an autocrine loop indicates thatinhibition of c-fms activity can have an impact on tumor associatedmacrophages but also on the tumor cells. Thus, in one embodiment, tumorsthat have an autocrine loop are targeted as the primary target. In otherembodiments, both TAMs and the tumor are targeted for a combined effect.In still other embodiments, tumors using a paracrine loop or anautocrine and paracrine loop are targeted.

The human c-fms antigen binding proteins provided herein can in certainembodiments be administered alone but can also be used in combinationwith one or more other cancer treatment options, such as, for example,chemotherapy, radiation therapy, or surgery. If administered with achemotherapeutic, the antigen binding protein can be administered beforeor after the chemotherapeutic agent or at the same time (e.g., as partof the same composition).

Chemotherapy treatments that can be used in combination with the antigenbinding proteins that are provided include, but are not limited to,anti-neoplastic agents including alkylating agents including: nitrogenmustards, such as mechlorethamine, cyclophosphamide, ifosfamide,melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU),lomustine (CCNU), and semustine (methyl-CCNU); Temodal™ (temozolamide),ethylenimines/methylmelamine such as thriethylenemelamine (TEM),triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM,altretamine); alkyl sulfonates such as busulfan; triazines such asdacarbazine (DTIC); antimetabolites including folic acid analogs such asmethotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil(5FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC,cytarabine), 5-azacytidine, 2,2′-difluorodeoxycytidine, purine analogssuch as 6-mercaptopurine, 6-thioguanine, azathioprine,2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA),fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA);natural products including antimitotic drugs such as paclitaxel, vincaalkaloids including vinblastine (VLB), vincristine, and vinorelbine,taxotere, estramustine, and estramustine phosphate; pipodophylotoxinssuch as etoposide and teniposide; antibiotics such as actimomycin D,daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin,bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin;enzymes such as L-asparaginase; biological response modifiers such asinterferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents includingplatinum coordination complexes such as cisplatin and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; Gemzar™ (gemcitabine), progestinsuch as hydroxyprogesterone caproate, medroxyprogesterone acetate andmegestrol acetate; estrogen such as diethylstilbestrol and ethinylestradiol equivalents; antiestrogen such as tamoxifen; androgensincluding testosterone propionate and fluoxymesterone/equivalents;antiandrogens such as flutamide, gonadotropin-releasing hormone analogsand leuprolide; and non-steroidal antiandrogens such as flutamide.Therapies targeting epigenetic mechanism including, but not limited to,histone deacetylase inhibitors, demethylating agents (e.g., Vidaza) andrelease of transcriptional repression (ATRA) therapies can also becombined with the antigen binding proteins.

Cancer therapies, which may be administered with an antigen bindingprotein, also include, but are not limited to, targeted therapies.Examples of targeted therapies include, but are not limited to, use oftherapeutic antibodies. Exemplary therapeutic antibodies, include, butare not limited to, mouse, mouse-human chimeric, CDR-grafted, humanizedand fully human antibodies, and synthetic antibodies, including, but notlimited to, those selected by screening antibody libraries. Exemplaryantibodies include, but are not limited to, those which bind to cellsurface proteins Her2, CDC20, CDC33, mucin-like glycoprotein, andepidermal growth factor receptor (EGFR) present on tumor cells, andoptionally induce a cytostatic and/or cytotoxic effect on tumor cellsdisplaying these proteins. Exemplary antibodies also include HERCEPTIN™(trastuzumab), which may be used to treat breast cancer and other formsof cancer, and RITUXAN™ (rituximab), ZEVALIN™ (ibritumomab tiuxetan),GLEEVEC™, and LYMPHOCIDE™ (epratuzumab), which may be used to treatnon-Hodgkin's lymphoma and other forms of cancer. Certain exemplaryantibodies also include ERBITUX™ (IMC-C225); ertinolib (Iressa); BEXXAR™(iodine 131 tositumomab); KDR (kinase domain receptor) inhibitors; antiVEGF antibodies and antagonists (e.g., Avastin™ and VEGAF-TRAP); antiVEGF receptor antibodies and antigen binding regions; anti-Ang-1 andAng-2 antibodies and antigen binding regions; antibodies to Tie-2 andother Ang-1 and Ang-2 receptors; Tie-2 ligands; antibodies against Tie-2kinase inhibitors; inhibitors of Hif-1a, and Campath™ (Alemtuzumab). Incertain embodiments, cancer therapy agents are polypeptides whichselectively induce apoptosis in tumor cells, including, but not limitedto, the TNF-related polypeptide TRAIL.

Additional specific examples of chemotherapeutic agents include, taxol,taxenes (e.g., docetaxel and Taxotere), modified paclitaxel (e.g.,Abraxane and Opaxio) doxorubicin, Avastin®, Sutent, Nexavar, and othermultikinase inhibitors, cisplatin and carboplatin, etoposide,gemcitabine, and vinblastine. Specific inhibitors of other kinases canalso be used in combination with the antigen binding proteins, includingbut not limited to, MAPK pathway inhibitors (e.g., inhibitors of ERK,JNK and p38), PI3kinase/AKT inhibitors and Pim inhibitors. Otherinhibitors include Hsp90 inhibitors, proteasome inhibitors (e.g.,Velcade) and multiple mechanism of action inhibitors such as Trisenox.

In certain embodiment, an antigen binding protein as provided herein isused in combination with one or more anti-angiogenic agents thatdecrease angiogenesis. Certain such agents include, but are not limitedto, IL-8 antagonists; Campath, B-FGF; FGF antagonists; Tek antagonists(Cerretti et al., U.S. Publication No. 2003/0162712; Cerretti et al.,U.S. Pat. No. 6,413,932, and Cerretti et al., U.S. Pat. No. 6,521,424,each of which is incorporated herein by reference for all purposes);anti-TWEAK agents (which include, but are not limited to, antibodies andantigen binding regions); soluble TWEAK receptor antagonists (Wiley,U.S. Pat. No. 6,727,225); an ADAM distintegrin domain to antagonize thebinding of integrin to its ligands (Fanslow et al., U.S. Publication No.2002/0042368); anti-eph receptor and anti-ephrin antibodies; antigenbinding regions, or antagonists (U.S. Pat. Nos. 5,981,245; 5,728,813;5,969,110; 6,596,852; 6,232,447; 6,057,124 and patent family membersthereof); anti-VEGF agents (e.g., antibodies or antigen binding regionsthat specifically bind VEGF, or soluble VEGF receptors or a ligandbinding regions thereof) such as Avastin™ or VEGF-TRAP™, and anti-VEGFreceptor agents (e.g., antibodies or antigen binding regions thatspecifically bind thereto), EGFR inhibitory agents (e.g., antibodies orantigen binding regions that specifically bind thereto) such aspanitumumab, IRESSA™ (gefitinib), TARCEVA™ (erlotinib), anti-Ang-1 andanti-Ang-2 agents (e.g., antibodies or antigen binding regionsspecifically binding thereto or to their receptors, e.g., Tie-2/TEK),and anti-Tie-2 kinase inhibitory agents (e.g., antibodies or antigenbinding regions that specifically bind and inhibit the activity ofgrowth factors, such as antagonists of hepatocyte growth factor (HGF,also known as Scatter Factor), and antibodies or antigen binding regionsthat specifically bind its receptor “c-met”; anti-PDGF-BB antagonists;antibodies and antigen binding regions to PDGF-BB ligands; and PDGFRkinase inhibitors.

Other anti-angiogenic agents that can be used in combination with anantigen binding protein include agents such as MMP-2(matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase9) inhibitors, and COX-II (cyclooxygenase II) inhibitors. Examples ofuseful COX-II inhibitors include CELEBREX™ (celecoxib), valdecoxib, androfecoxib.

In certain embodiments, cancer therapy agents are angiogenesisinhibitors. Certain such inhibitors include, but are not limited to,SD-7784 (Pfizer, USA); cilengitide. (Merck KGaA, Germany, EPO 770622);pegaptanib octasodium, (Gilead Sciences, USA); Alphastatin, (BioActa,UK); M-PGA, (Celgene, USA, U.S. Pat. No. 5,712,291); ilomastat, (Arriva,USA, U.S. Pat. No. 5,892,112); semaxanib, (Pfizer, USA, U.S. Pat. No.5,792,783); vatalanib, (Novartis, Switzerland); 2-methoxyestradiol,(EntreMed, USA); TLC ELL-12, (Elan, Ireland); anecortave acetate,(Alcon, USA); alpha-D148 Mab, (Amgen, USA); CEP-7055, (Cephalon, USA);anti-Vn Mab, (Crucell, Netherlands) DAC:antiangiogenic, (ConjuChem,Canada); Angiocidin, (InKine Pharmaceutical, USA); KM-2550, (KyowaHakko, Japan); SU-0879, (Pfizer, USA); CGP-79787, (Novartis,Switzerland, EP 970070); ARGENT technology, (Ariad, USA); YIGSR-Stealth,(Johnson & Johnson, USA); fibrinogen-E fragment, (BioActa, UK);angiogenesis inhibitor, (Trigen, UK); TBC-1635, (EncysivePharmaceuticals, USA); SC-236, (Pfizer, USA); ABT-567, (Abbott, USA);Metastatin, (EntreMed, USA); angiogenesis inhibitor, (Tripep, Sweden);maspin, (Sosei, Japan); 2-methoxyestradiol, (Oncology SciencesCorporation, USA); ER-68203-00, (IVAX, USA); Benefin, (Lane Labs, USA);Tz-93, (Tsumura, Japan); TAN-1120, (Takeda, Japan); FR-111142,(Fujisawa, Japan, JP 02233610); platelet factor 4, (RepliGen, USA, EP407122); vascular endothelial growth factor antagonist, (Borean,Denmark); cancer therapy, (University of South Carolina, USA);bevacizumab (pINN), (Genentech, USA); angiogenesis inhibitors, (SUGEN,USA); XL 784, (Exelixis, USA); XL 647, (Exelixis, USA); MAb, alpha5beta3integrin, second generation, (Applied Molecular Evolution, USA andMedImmune, USA); gene therapy, retinopathy, (Oxford BioMedica, UK);enzastaurin hydrochloride (USAN), (Lilly, USA); CEP 7055, (Cephalon, USAand Sanofi-Synthelabo, France); BC 1, (Genoa Institute of CancerResearch, Italy); angiogenesis inhibitor, (Alchemia, Australia); VEGFantagonist, (Regeneron, USA); rBPI 21 and BPI-derived antiangiogenic,(XOMA, USA); PI 88, (Progen, Australia); cilengitide (pINN), (MerckKGaA, German; Munich Technical University, Germany, Scripps Clinic andResearch Foundation, USA); cetuximab (INN), (Aventis, France); AVE 8062,(Ajinomoto, Japan); AS 1404, (Cancer Research Laboratory, New Zealand);SG 292, (Telios, USA); Endostatin, (Boston Childrens Hospital, USA); ATN161, (Attenuon, USA); ANGIOSTATIN, (Boston Childrens Hospital, USA);2-methoxyestradiol, (Boston Childrens Hospital, USA); ZD 6474,(AstraZeneca, UK); ZD 6126, (Angiogene Pharmaceuticals, UK); PPI 2458,(Praecis, USA); AZD 9935, (AstraZeneca, UK); AZD 2171, (AstraZeneca,UK); vatalanib (pINN), (Novartis, Switzerland and Schering AG, Germany);tissue factor pathway inhibitors, (EntreMed, USA); pegaptanib (Pinn),(Gilead Sciences, USA); xanthorrhizol, (Yonsei University, South Korea);vaccine, gene-based, VEGF-2, (Scripps Clinic and Research Foundation,USA); SPV5.2, (Supratek, Canada); SDX 103, (University of California atSan Diego, USA); PX 478, (ProIX, USA); METASTATIN, (EntreMed, USA);troponin 1, (Harvard University, USA); SU 6668, (SUGEN, USA); OXI 4503,(OXiGENE, USA); o-guanidines, (Dimensional Pharmaceuticals, USA);motuporamine C, (British Columbia University, Canada); CDP 791,(Celltech Group, UK); atiprimod (PINN), (GlaxoSmithKline, UK); E 7820,(Eisai, Japan); CYC 381, (Harvard University, USA); AE 941, (Aeterna,Canada); vaccine, angiogenesis, (EntreMed, USA); urokinase plasminogenactivator inhibitor, (Dendreon, USA); oglufanide (pINN), (Melmotte,USA); HIF-1alfa inhibitors, (Xenova, UK); CEP 5214, (Cephalon, USA); BAYRES 2622, (Bayer, Germany); Angiocidin, (InKine, USA); A6, (Angstrom,USA); KR 31372, (Korea Research Institute of Chemical Technology, SouthKorea); GW 2286, (GlaxoSmithKline, UK); EHT 0101, (ExonHit, France); CP868596, (Pfizer, USA); CP 564959, (OSI, USA); CP 547632, (Pfizer, USA);786034, (GlaxoSmithKline, UK); KRN 633, (Kirin Brewery, Japan); drugdelivery system, intraocular, 2-methoxyestradiol, (EntreMed, USA);anginex, (Maastricht University, Netherlands, and Minnesota University,USA); ABT 510, (Abbott, USA); ML 993, (Novartis, Switzerland); VEGI,(Proteom Tech, USA); tumor necrosis factor-alpha inhibitors, (NationalInstitute on Aging, USA); SU 11248, (Pfizer, USA and SUGEN USA); ABT518, (Abbott, USA); YH16, (Yantai Rongchang, China); S-3APG, (BostonChildrens Hospital, USA and EntreMed, USA); MAb, KDR, (ImClone Systems,USA); MAb, alpha5 beta1, (Protein Design, USA); KDR kinase inhibitor,(Celltech Group, UK, and Johnson & Johnson, USA); GFB 116, (SouthFlorida University, USA and Yale University, USA); CS 706, (Sankyo,Japan); combretastatin A4 prodrug, (Arizona State University, USA);chondroitinase AC, (IBEX, Canada); BAY RES 2690, (Bayer, Germany); AGM1470, (Harvard University, USA, Takeda, Japan, and TAP, USA); AG 13925,(Agouron, USA); Tetrathiomolybdate, (University of Michigan, USA); GCS100, (Wayne State University, USA) CV 247, (Ivy Medical, UK); CKD 732,(Chong Kun Dang, South Korea); MAb, vascular endothelium growth factor,(Xenova, UK); irsogladine (INN), (Nippon Shinyaku, Japan); RG 13577,(Aventis, France); WX 360, (Wilex, Germany); squalamine (pINN),(Genaera, USA); RPI 4610, (Sirna, USA); cancer therapy, (Marinova,Australia); heparanase inhibitors, (InSight, Israel); KL 3106, (Kolon,South Korea); Honokiol, (Emory University, USA); ZK CDK, (Schering AG,Germany); ZK Angio, (Schering AG, Germany); ZK 229561, (Novartis,Switzerland, and Schering AG, Germany); XMP 300, (XOMA, USA); VGA 1102,(Taisho, Japan); VEGF receptor modulators, (Pharmacopeia, USA);VE-cadherin-2 antagonists, (ImClone Systems, USA); Vasostatin, (NationalInstitutes of Health, USA); vaccine, Flk-1, (ImClone Systems, USA); TZ93, (Tsumura, Japan); TumStatin, (Beth Israel Hospital, USA); truncatedsoluble FLT 1 (vascular endothelial growth factor receptor 1), (Merck &Co, USA); Tie-2 ligands, (Regeneron, USA); thrombospondin 1 inhibitor,(Allegheny Health, Education and Research Foundation, USA);2-Benzenesulfonamide,4-(5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)-; Arriva; andC-Met. AVE 8062((2S)-2-amino-3-hydroxy-N-[2-methoxy-5-[(1Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl]propanamidemonohydrochloride); metelimumab (pINN)(immunoglobulin G4, anti-(humantransforming growth factor .beta.1 (human monoclonal CAT192.gamma.4-chain)), disulfide with human monoclonal CAT192.kappa.-chain dimer); Flt3 ligand; CD40 ligand; interleukin-2;interleukin-12; 4-1BB ligand; anti-4-1BB antibodies; TNF antagonists andTNF receptor antagonists including TNFR/Fc, TWEAK antagonists andTWEAK-R antagonists including TWEAK-R/Fc; TRAIL; VEGF antagonistsincluding anti-VEGF antibodies; VEGF receptor (including VEGF-R1 andVEGF-R2, also known as Flt1 and Flk1 or KDR) antagonists; CD148 (alsoreferred to as DEP-1, ECRTP, and PTPRJ, see Takahashi et al., J. Am.Soc. Nephrol. 10: 213545 (1999), hereby incorporated by reference forany purpose) agonists; thrombospondin 1 inhibitor, and inhibitors of oneor both of Tie-2 or Tie-2 ligands (such as Ang-2). A number ofinhibitors of Ang-2 are known in the art, including anti-Ang-2antibodies described in published U.S. Patent Application No.20030124129 (corresponding to PCT Application No. WO03/030833), and U.S.Pat. No. 6,166,185, the contents of which are hereby incorporated byreference in their entirety. Additionally, Ang-2 peptibodies are alsoknown in the art, and can be found in, for example, published U.S.Patent Application No. 20030229023 (corresponding to PCT Application No.WO03/057134), and published U.S. Patent Application No. 20030236193, thecontents of which are hereby incorporated by reference in their entiretyfor all purposes.

Certain cancer therapy agents include, but are not limited to:thalidomide and thalidomide analogues(N-(2,6-dioxo-3-piperidyl)phthalimide); tecogalan sodium (sulfatedpolysaccharide peptidoglycan); TAN 1120(8-acetyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-10-[[octahydro-5-hydroxy-2-(2-hydroxypropyl)-4,10-dimethylpyrano[3,4-d]-1,3,6-dioxazocin-8-yl]oxy]-5,12-naphthacenedione);suradista(7,7′-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3-naphthalenedisulfonic acid tetrasodium salt); SU 302; SU 301; SU 1498((E)-2-cyano-3-[4-hydroxy-3,5-bis(1-methylethyl)phenyl]-N-(3-phenylpropyl)-2-propenamide);SU 1433 (4-(6,7-dimethyl-2-quinoxalinyl)-1,2-benzenediol); ST 1514; SR25989; soluble Tie-2; SERM derivatives, Pharmos; semaxanib(pINN)(3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one);S 836; RG 8803; RESTIN; R 440(3-(1-methyl-1H-indol-3-yl)-4-(1-methyl-6-nitro-1H-indol-3-yl)-1H-pyrrole-2,5-dione);R 123942(1-[6-(1,2,4-thiadiazol-5-yl)-3-pyridazinyl]-N-[3-(trifluoromethyl)phenyl]-4-piperidinamine);prolyl hydroxylase inhibitor; progression elevated genes; prinomastat(INN)((S)-2,2-dimethyl-4-[[p-(4-pyridyloxy)phenyl]sulphonyl]-3-thiomorpholinecarbohydroxamicacid); NV 1030; NM 3(8-hydroxy-6-methoxy-alpha-methyl-1-oxo-1H-2-benzopyran-3-acetic acid);NF 681; NF 050; MIG; METH 2; METH 1; manassantin B(alpha-[1-[4-[5-[4-[2-(3,4-dimethoxyphenyl)-2-hydroxy-1-methylethoxy]-3-methoxyphenyl]tetrahydro-3,4-dimethyl-2-furanyl]-2-methoxyphenoxy]ethyl]-1,3-benzodioxole-5-methanol);KDR monoclonal antibody; alpha5beta3 integrin monoclonal antibody; LY290293 (2-amino-4-(3-pyridinyl)-4H-naphtho[1,2-b]pyran-3-carbonitrile);KP 0201448; KM 2550; integrin-specific peptides; INGN 401; GYKI 66475;GYKI 66462; greenstatin (101-354-plasminogen (human)); gene therapy forrheumatoid arthritis, prostate cancer, ovarian cancer, glioma,endostatin, colorectal cancer, ATF BTPI, antiangiogenesis genes,angiogenesis inhibitor, or angiogenesis; gelatinase inhibitor, FR 111142(4,5-dihydroxy-2-hexenoic acid5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2.5]oct-6-ylester); forfenimex (PINN)(S)-alpha-amino-3-hydroxy-4-(hydroxymethyl)benzeneacetic acid);fibronectin antagonist(1-acetyl-L-prolyl-L-histidyl-L-seryl-L-cysteinyl-L-aspartamide);fibroblast growth factor receptor inhibitor; fibroblast growth factorantagonist; FCE 27164(7,7′-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3,5-naphthalenetrisulfonicacid hexasodium salt); FCE 26752(8,8′-[carbonylbis[imino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino(1-methyl-1H-pyrrole-4,2-diyl)carbonylimino]]bis-1,3,6-naphthalenetrisulfonicacid); endothelial monocyte activating polypeptide II; VEGFR antisenseoligonucleotide; anti-angiogenic and trophic factors; ANCHOR angiostaticagent; endostatin; Del-1 angiogenic protein; CT 3577; contortrostatin;CM 101; chondroitinase AC; CDP 845; CanStatin; BST 2002; BST 2001; BLS0597; BIBF 1000; ARRESTIN; apomigren (1304-1388-type XV collagen (humangene COL15A1 alpha1-chain precursor)); angioinhibin; aaATIII; A 36;9alpha-fluoromedroxyprogesterone acetate((6-alpha)-17-(acetyloxy)-9-fluoro-6-methyl-pregn-4-ene-3,20-dione);2-methyl-2-phthalimidino-glutaric acid(2-(1,3-dihydro-1-oxo-2H-isoindol-2-yl)-2-methylpentanedioic acid);Yttrium 90 labelled monoclonal antibody BC-1; Semaxanib(3-(4,5-Dimethylpyrrol-2-ylmethylene)indolin-2-one) (C15H14N2O); PI 88(phosphomannopentaose sulfate); Alvocidib(4H-1-Benzopyran-4-one,2-(2-chlorophenyl)-5,7-dihydroxy-8-(3-hydroxy-1-methyl-4-piperidinyl)-cis-(−)-)(C21-H20 Cl N O5); E 7820; SU 11248(5-[3-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide) (C22H27FN4O2); Squalamine(Cholestane-7,24-diol, 3-[[3-[(4-aminobutyl)aminopropyl]amino]-,24-(hydrogen sulfate), (3.beta.,5.alpha.,7.alpha.)-) (C34H65N3O.sub.5S);Eriochrome Black T; AGM 1470 (Carbamic acid, (chloroacetyl)-,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-ylester, [3R-[3alpha,4alpha(2R,3R),5beta,6beta]]) (C19H28ClNO6); AZD 9935;BIBF 1000; AZD 2171; ABT 828; KS-interleukin-2; Uteroglobin; A 6; NSC639366(1-[3-(Diethylamino)-2-hydroxypropylamino]-4-(oxyran-2-ylmethylamino)anthraquinonefumerate) (C24H29N3O4.C4H4O4); ISV 616; anti-ED-B fusion proteins; HUI77; Troponin I; BC-1 monoclonal antibody; SPV 5.2; ER 68203; CKD 731(3-(3,4,5-Trimethoxyphenyl)-2(E)-propenoic acid (3R,4S,5S,6R)-4-[2(R)-methyl-3(R)-3(R)-(3-methyl-2-butenyl)oxiran-2-yl]-5-methoxy-1-oxaspiro[2.5]oct-6-ylester) (C28H38O8); IMC-1C11; aaATIII; SC 7; CM 101; Angiocol; Kringle 5;CKD 732 (3-[4-[2-(Dimethylamino)ethoxy]phenyl]-2(E)-propenoic acid)(C29H41NO6); U 995; Canstatin; SQ 885; CT 2584(1-[11-(Dodecylamino)-10-hydroxyundecyl]-3,7-dimethylxanthine) (C30 H55N5 O3); Salmosin; EMAP II; TX 1920(1-(4-Methylpiperazino)-2-(2-nitro-1H-1-imidazoyl)-1-ethanone)(C10H15N5O3); Alpha-v Beta-x inhibitor; CHIR 11509(N-(1-Propynyl)glycyl-[N-(2-naphthyl)]glycyl-[N-(carbamoylmethyl)]glycinebis(4-methoxyphenyl)methylamide) (C36H37N5O6); BST 2002; BST 2001; B0829; FR 111142; 4,5-Dihydroxy-2(E)-hexenoic acid(3R,4S,5S,6R)-4-[1(R),2(R)-epoxy-1,5-dimethyl-4-hexenyl]-5-methoxy-1-oxaspiro[2.5]octan-6-ylester (C22H34O7); and kinase inhibitors including, but not limited to,N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-1-phthalazinamine;4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-2-pyridinecarboxamide;N-[2-(diethylamino)ethyl]-5-[(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide;3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl)butyl]amino]carbonyl]amino]-4-isothiazolecarboxamide;N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl)methoxy]-4-quinazolinamine;3-[5,6,7,13-tetrahydro-9-[(1-methylethoxy)methyl]-5-oxo-12H-indeno[2,1-a]pyrrolo[3,4-c]carbazol-12-yl]propylester N,N-dimethyl-glycine;N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide;N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[[[2-(methylsulfonyl)ethyl]amino]methyl]-2-furanyl]4-quinazolinamine;4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide;N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine;N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine;N-(3-((((2R)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-2-(3-(1,3-oxazol-5-yl)phenyl)amino)-3-pyridinecarboxamide;2-(((4-fluorophenyl)methyl)amino)-N-(3-((((2R)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-3-pyridinecarboxamide;N-[3-(Azetidin-3-ylmethoxy)-5-trifluoromethyl-phenyl]-2-(4-fluoro-benzylamino)-nicotinamide;6-fluoro-N-(4-(1-methylethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;2-((4-pyridinylmethyl)amino)-N-(3-(((2S)-2-pyrrolidinylmethyl)oxy)-5-(trifluoromethyl)phenyl)-3-pyridinecarboxamide;N-(3-(1,1-dimethylethyl)-1H-pyrazol-5-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(3,3-dimethyl-2,3-dihydro-1-benzofuran-6-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(3-((((2S)-1-methyl-2-pyrrolidinyl)methyl)oxy)-5-(trifluoromethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;2-((4-pyridinylmethyl)amino)-N-(3-((2-(1-pyrrolidinyl)ethyl)oxy)-4-(trifluoromethyl)phenyl)-3-pyridinecarboxamide;N-(3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(4-(pentafluoroethyl)-3-(((2S)-2-pyrrolidinylmethyl)oxy)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(3-((3-azetidinylmethyl)oxy)-5-(trifluoromethyl)phenyl)-2-((4-pyridinylmethyl)amino)-3-pyridinecarboxamide;N-(3-(4-piperidinyloxy)-5-(trifluoromethyl)phenyl)-2-((2-(3-pyridinyl)ethyl)amino)-3-pyridinecarboxamide;N-(4,4-dimethyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-2-(1H-indazol-6-ylamino)-nicotinamide;2-(1H-indazol-6-ylamino)-N-[3-(1-methylpyrrolidin-2-ylmethoxy)-5-trifluoromethyl-phenyl]-nicotinamide;N-[1-(2-dimethylamino-acetyl)-3,3-dimethyl-2,3-dihydro-1H-indol-6-yl]-2-(1H-indazol-6-ylamino)-nicotinamide;2-(1H-indazol-6-ylamino)-N-[3-(pyrrolidin-2-ylmethoxy)-5-trifluoromethyl-phenyl]-nicotinamide;N-(1-acetyl-3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-(1H-indazol-6-ylamino)-nicotinamide;N-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydro-isoquinolin-7-yl)-2-(1H-indazol-6-ylamino)-nicotinamide;N-[4-(tert-butyl)-3-(3-piperidylpropyl)phenyl][2-(1H-indazol-6-ylamino)(3-pyridyl)]carboxamide;N-[5-(tert-butyl)isoxazol-3-yl][2-(1H-indazol-6-ylamino)(3-pyridyl)]carboxamide;andN-[4-(tert-butyl)phenyl][2-(1H-indazol-6-ylamino)(3-pyridyl)]carboxamide,and kinase inhibitors disclosed in U.S. Pat. Nos. 6,258,812; 6,235,764;6,630,500; 6,515,004; 6,713,485; 5,521,184; 5,770,599; 5,747,498;5,990,141; U.S. Publication No. U.S. 20030105091; and Patent CooperationTreaty publication nos. WO 01/37820; WO 01/32651; WO 02/68406; WO02/66470; WO 02/55501; WO 04/05279; WO 04/07481; WO 04/07458; WO04/09784; WO 02/59110; WO 99/45009; WO 98/35958; WO 00/59509; WO99/61422; WO 00/12089; and WO 00/02871, each of which publications arehereby incorporated by reference for all purposes.

An antigen binding protein as provided herein can also be used incombination with a growth factor inhibitor. Examples of such agents,include, but are not limited to, agents that can inhibit EGF-R(epidermal growth factor receptor) responses, such as EGF-R antibodies,EGF antibodies, and molecules that are EGF-R inhibitors; VEGF (vascularendothelial growth factor) inhibitors, such as VEGF receptors andmolecules that can inhibit VEGF; and erbB2 receptor inhibitors, such asorganic molecules or antibodies that bind to the erbB2 receptor, forexample, HERCEPTIN™ (Genentech, Inc.). EGF-R inhibitors are describedin, for example in U.S. Pat. No. 5,747,498, WO 98/14451, WO 95/19970,and WO 98/02434.

Specific examples of combination therapies include, for instance, thec-fms antigen binding protein with taxol or taxanes (e.g., docetaxel orTaxotere) or a modified paclitaxel (e.g., Abraxane or Opaxio),doxorubicin and/or Avastin® for the treatment of breast cancer; thehuman c-fms antigen binding protein with a multi-kinase inhibitor, MKI,(Sutent, Nexavar, or 706) and/or doxorubicin for treatment of kidneycancer; the c-fms antigen binding protein with cisplatin and/orradiation for the treatment of squamous cell carcinoma; the c-fmsantigen binding protein with taxol and/or carboplatin for the treatmentof lung cancer.

In addition to the applications in oncology, the binding proteinsprovided herein can be used in the treatment or detection ofinflammatory diseases. In those inflammatory diseases where macrophagescontribute to the pathology of disease, the ability of the c-fms antigenbinding protein to reduce levels of macrophages in other cellularcompartments indicates a useful role in treating these diseases. Severalstudies suggest that human c-fms antigen binding protein may play a rolein the modulation of inflammatory diseases, like, for example,inflammatory arthritis, atherosclerosis, and multiple sclerosis.

Additional diseases that can be treated include, but are not limited to,inflammatory bowel disease, Crohn's disease, ulcerative colitis,rheumatoid spondylitis, ankylosing spondylitis, arthritis, psoriaticarthritis, rheumatoid arthritis, osteoarthritis, eczema, contactdermatitis, psoriasis, toxic shock syndrome, sepsis, septic shock,endotoxic shock, asthma, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoidosis, osteoporosis, restenosis, cardiac andrenal reperfusion injury, thrombosis, glomerularonephritis, diabetes,graft vs. host reaction, allograft rejection, multiple sclerosis, muscledegeneration, muscular dystrophy, Alzheimer's disease and stroke.

The antigen binding proteins can also be used to treat cachexia becausethe proinflammatory cytokines produced by macrophages are considered tobe involved in cachexia pathology (Sweet et al., 2002, J. Immunol.168:392-399; Boddaert et al., 2006, Curr. Opin. Oncol. 8:335-340 andWang et al. 2006 J. Endocrinology 190:415-423).

Given the ability of the antigen binding proteins to be used to treatvarious inflammatory diseases, they can be used or combined with variousother anti-inflammatory agents. Examples of such agents include, but arenot limited to TNF-alpha inhibitors such as TNF drugs (e.g., HUMIRA™,REMICADE™) and TNF receptor immunoglobulin molecules (such as ENBREL™),IL-1 inhibitors, receptor antagonists or soluble IL-1ra (e.g. Kineret orICE inhibitors), COX-2 inhibitors and metalloprotease inhibitors such asthose described above, and alpha-2-delta ligands (e.g., PREGABALIN™ andNEUROTIN™).

In certain embodiments, antigen binding proteins can also be used totreat various bone diseases in view of the important role of c-fms inosteoclast development and activation (e.g., Rolf, F. et al. (2008) J.Biol. Chem. 55:340-349, and Watarn, A. et al. (2006) J. Bone MineralMetabolism 24:274-282). The antigen binding proteins can thus be usefulfor treating patients suffering from various medical disorders thatinvolve excessive bone loss or patients who require the formation of newbone even where there may not necessarily be excessive osteoclastactivity. Excessive osteoclast activity is associated with numerousosteopenic disorders that can be treated with the antigen bindingproteins that are provided, including ostopenia, osteoporosis,periodontitis, Paget's disease, bone loss due to immobilization, lyticbone metastases and arthritis, including rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis and other conditions that involve boneerosion. Some cancers are known to increase osteoclast activity andinduce bone resorption, such as breast and prostate cancer. Multiplemyeloma, which arises in bone marrow, also is associated with bone loss.

With respect to bone metastases of cancer, inhibition of the CSF-1/c-fmsaxis through the use of the antigen binding proteins provided hereincould be of therapeutic benefit through multiple mechanisms of action.These would include inhibition of invasion and metastasis through lossof the matrix degrading enzymes produced by TAMs, interference withtumor cell seeding within bone marrow through loss of osteoclast numbersand function, inhibition of metastatic tumor growth through previouslymentioned reduction of TAMs and inhibition of bone osteolysis associatedwith bone metastatic lesions (Ohno, H. et al. (2008) Molecular CancerTherapeutics. 5:2634-2643). The antigen binding proteins can also havetherapeutic benefit for osteosarcoma, which is a cancer of the bone.

Various other low bone mass conditions can also be treated including avariety of forms of osteoporosis, including but not limited to,glucocorticoid induced osteoporosis, osteoporosis induced aftertransplantation, osteoporosis associated with chemotherapy (i.e.,chemotherapy induced osteoporosis), immobilization induced osteoporosis,osteoporosis due to mechanical unloading, and osteoporosis associatedwith anticonvulsant use. Additional bone diseases that can be treatedinclude bone disease associated with renal failure and nutritional,gastrointestinal and/or hepatic associated bone diseases.

Different forms of arthritis can also be treated, examples includingosteoarthritis and rheumatoid arthritis. The antigen binding proteinscan also be used to treat systemic bone loss associated with arthritis(e.g., rheumatoid arthritis). In treating arthritis, patients maybenefit by perilesional or intralesional injections of the subjectantigen binding proteins. For example, the antigen binding protein canbe injected adjacent to or directly into an inflamed joint, thusstimulating repair of damaged bone at the site.

The antigen binding proteins described herein can also be used invarious bone repair applications. For example, they can be useful inretarding wear debris osteolysis associated with artificial joints,accelerating the repair of bone fractures, and enhancing theincorporation of bone grafts into the surrounding living bone into whichthey have been engrafted.

The antigen binding proteins provided herein when used to treat bonedisorders can be administered alone or in combination with othertherapeutic agents, for example, in combination with cancer therapyagents, with agents that inhibit osteoclast activity or with otheragents that enhance osteoblast activity. For example, the antigenbinding proteins can be administered to cancer patients undergoingradiation therapy or chemotherapy. Chemotherapies used in combinationwith the antigen binding proteins may include anthracyclines, taxol,tamoxifene, doxorubicin, 5-fluorouracil, oxaloplatin, Velcade®([(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronicacid) and/or other small molecule drugs that are used in treatingcancer.

The antigen binding proteins can be used alone for the treatment of theabove referenced conditions resulting in loss of bone mass or incombination with a therapeutically effective amount of a bone growthpromoting (anabolic) agent or a bone anti-resorptive agent including butnot limited to: bone morphogenic factors designated BMP-1 to BMP-12;transforming growth factor-β and TGF-β family members; fibroblast growthfactors FGF-1 to FGF-10; interleukin-1 inhibitors (including IL-1ra,antibodies to IL-1 and antibodies to IL-1 receptors); TNFα inhibitors(including etanercept, adalibumab and infliximab); RANK ligandinhibitors (including soluble RANK, osteoprotegerin and antagonisticantibodies that specifically bind RANK or RANK ligand), Dkk-1 inhibitors(e.g., anti-Dkk-1 antibodies) parathyroid hormone, E seriesprostaglandins, bisphosphonates and bone-enhancing minerals such asfluoride and calcium. Anabolic agents that can be used in combinationwith the antigen binding proteins and functional fragments thereofinclude parathyroid hormone and insulin-like growth factor (IGF),wherein the latter agent is preferably complexed with an IGF bindingprotein. An IL-1 receptor antagonist suitable for such combinationtreatment is described in WO89/11540 and a suitable soluble TNFreceptor-1 is described in WO98/01555. Exemplary RANK ligand antagonistsare disclosed, for example, in WO 03/086289, WO 03/002713, U.S. Pat.Nos. 6,740,511 and 6,479,635. All of the aforementioned patents andpatent applications are hereby incorporated by reference.

The antigen binding proteins can also be used to inhibit angiogenesis(e.g., in tumors). For example, the antigen binding proteins can be usedto decrease blood vessel formation in cases where inflammatoryangiogenesis is driven primarily by FGF-2. In some embodiments, theantigen binding proteins are used to inhibit angiogenesis in tumorswhere VEGF levels are low and tumor vascular density is high.

Diagnostic Methods

The antigen binding proteins of the described can be used for diagnosticpurposes to detect, diagnose, or monitor diseases and/or conditionsassociated with c-fms. The disclosed provides for the detection of thepresence of c-fms in a sample using classical immunohistological methodsknown to those of skill in the art (e.g., Tijssen, 1993, Practice andTheory of Enzyme Immunoassays, Vol 15 (Eds R. H. Burdon and P. H. vanKnippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al.,1985, J. Cell. Biol. 101:976-985; Jalkanen et al., 1987, J. Cell Biol.105:3087-3096). The detection of c-fms can be performed in vivo or invitro.

Diagnostic applications provided herein include use of the antigenbinding proteins to detect expression of c-fms and binding of theligands to c-fms. Examples of methods useful in the detection of thepresence of c-fms include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA).

For diagnostic applications, the antigen binding protein typically willbe labeled with a detectable labeling group. Suitable labeling groupsinclude, but are not limited to, the following: radioisotopes orradionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I),fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic groups (e.g., horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase), chemiluminescent groups, biotinylgroups, or predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags). In someembodiments, the labeling group is coupled to the antigen bindingprotein via spacer arms of various lengths to reduce potential sterichindrance. Various methods for labeling proteins are known in the artand may be used.

In another aspect, an antigen binding protein can be used to identify acell or cells that express c-fms. In a specific embodiment, the antigenbinding protein is labeled with a labeling group and the binding of thelabeled antigen binding protein to c-fms is detected. In a furtherspecific embodiment, the binding of the antigen binding protein to c-fmsdetected in vivo. In a further specific embodiment, the c-fms antigenbinding protein is isolated and measured using techniques known in theart. See, for example, Harlow and Lane, 1988, Antibodies: A LaboratoryManual, New York: Cold Spring Harbor (ed. 1991 and periodicsupplements); John E. Coligan, ed., 1993, Current Protocols InImmunology New York: John Wiley & Sons.

Another aspect of the disclosed provides for detecting the presence of atest molecule that competes for binding to c-fms with the antigenbinding proteins provided. An example of one such assay would involvedetecting the amount of free antigen binding protein in a solutioncontaining an amount of c-fms in the presence or absence of the testmolecule. An increase in the amount of free antigen binding protein(i.e., the antigen binding protein not bound to c-fms) would indicatethat the test molecule is capable of competing for c-fms binding withthe antigen binding protein. In one embodiment, the antigen bindingprotein is labeled with a labeling group. Alternatively, the testmolecule is labeled and the amount of free test molecule is monitored inthe presence and absence of an antigen binding protein.

Methods of Treatment: Pharmaceutical Formulations, Routes ofAdministration

Methods of using the antigen binding proteins are also provided. In somemethods, an antigen binding protein is provided to a patient. Theantigen binding protein inhibits binding of CSF-1 to human c-fms. Theadministration of an antigen binding protein in some methods can alsoinhibit autophosphorylation of human c-fms by inhibiting binding ofCSF-1 to human c-fms. Further, in certain methods, monocyte chemotaxisis reduced by administering an effective amount of at least one antigenbinding protein to a patient. Monocyte migration into tumors in somemethods is inhibited by administering an effective amount of an antigenbinding protein. In addition, the accumulation of tumor associatedmacrophage in a tumor or diseased tissue can be inhibited byadministering an antigen binding protein as provided herein.

Pharmaceutical compositions that comprise a therapeutically effectiveamount of one or a plurality of the antigen binding proteins and apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative, and/or adjuvant are also provided. In addition, methods oftreating a patient by administering such pharmaceutical composition areincluded. The term “patient” includes human patients.

Acceptable formulation materials are nontoxic to recipients at thedosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of human c-fms antigen binding proteins are provided.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantigen binding proteins disclosed. In certain embodiments, the primaryvehicle or carrier in a pharmaceutical composition may be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier maybe water for injection, physiological saline solution or artificialcerebrospinal fluid, possibly supplemented with other materials commonin compositions for parenteral administration. Neutral buffered salineor saline mixed with serum albumin are further exemplary vehicles. Inspecific embodiments, pharmaceutical compositions comprise Tris bufferof about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and mayfurther include sorbitol or a suitable substitute. In certainembodiments, human c-fms antigen binding protein compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (REMINGTON'SPHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, the human c-fmsantigen binding protein may be formulated as a lyophilizate usingappropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery.Alternatively, the compositions may be selected for inhalation or fordelivery through the digestive tract, such as orally. Preparation ofsuch pharmaceutically acceptable compositions is within the skill of theart.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions may be provided in the form of a pyrogen-free, parenterallyacceptable aqueous solution comprising the desired human c-fms antigenbinding protein in a pharmaceutically acceptable vehicle. A particularlysuitable vehicle for parenteral injection is sterile distilled water inwhich the human c-fms antigen binding protein is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe product which can be delivered via depot injection. In certainembodiments, hyaluronic acid may also be used, having the effect ofpromoting sustained duration in the circulation. In certain embodiments,implantable drug delivery devices may be used to introduce the desiredantigen binding protein.

Certain pharmaceutical compositions are formulated for inhalation. Insome embodiments, human c-fms antigen binding proteins are formulated asa dry, inhalable powder. In specific embodiments, human c-fms antigenbinding protein inhalation solutions may also be formulated with apropellant for aerosol delivery. In certain embodiments, solutions maybe nebulized. Pulmonary administration and formulation methods thereforeare further described in International Patent Application No.PCT/US94/001875, which is incorporated by reference and describespulmonary delivery of chemically modified proteins. Some formulationscan be administered orally. Human c-fms antigen binding proteins thatare administered in this fashion can be formulated with or withoutcarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized Additional agents can be includedto facilitate absorption of the human c-fms antigen binding protein.Diluents, flavorings, low melting point waxes, vegetable oils,lubricants, suspending agents, tablet disintegrating agents, and bindersmay also be employed.

Some pharmaceutical compositions comprise an effective quantity of oneor a plurality of human c-fms antigen binding proteins in a mixture withnon-toxic excipients that are suitable for the manufacture of tablets.By dissolving the tablets in sterile water, or another appropriatevehicle, solutions may be prepared in unit-dose form. Suitableexcipients include, but are not limited to, inert diluents, such ascalcium carbonate, sodium carbonate or bicarbonate, lactose, or calciumphosphate; or binding agents, such as starch, gelatin, or acacia; orlubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving human c-fms antigen bindingproteins in sustained- or controlled-delivery formulations. Techniquesfor formulating a variety of other sustained- or controlled-deliverymeans, such as liposome carriers, bio-erodible microparticles or porousbeads and depot injections, are also known to those skilled in the art.See, for example, International Patent Application No. PCT/US93/00829,which is incorporated by reference and describes controlled release ofporous polymeric microparticles for delivery of pharmaceuticalcompositions. Sustained-release preparations may include semipermeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Sustained release matrices may include polyesters,hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 andEuropean Patent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., 1981, supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions may also includeliposomes that can be prepared by any of several methods known in theart. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A.82:3688-3692; European Patent Application Publication Nos. EP 036,676;EP 088,046 and EP 143,949, incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, cells expressing a recombinant antigen bindingprotein as disclosed herein is encapsulated for delivery (see, Invest.Ophthalmol Vis Sci 43:3292-3298, 2002 and Proc. Natl. Acad. Sciences103:3896-3901, 2006).

In certain formulations, an antigen binding protein has a concentrationof at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml,70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml or 150 mg/ml. Some formulationscontain a buffer, sucrose and polysorbate. An example of a formulationis one containing 50-100 mg/ml of antigen binding protein, 5-20 mMsodium acetate, 5-10% w/v sucrose, and 0.002-0.008% w/v polysorbate.Certain, formulations, for instance, contain 65-75 mg/ml of an antigenbinding protein in 9-11 mM sodium acetate buffer, 8-10% w/v sucrose, and0.005-0.006% w/v polysorbate. The pH of certain such formulations is inthe range of 4.5-6. Other formulations have a pH of 5.0-5.5 (e.g., pH of5.0, 5.2 or 5.4).

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. Kits for producing asingle-dose administration unit are also provided. Certain kits containa first container having a dried protein and a second container havingan aqueous formulation. In certain embodiments, kits containing singleand multi-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided. The therapeutically effective amount of ahuman c-fms antigen binding protein-containing pharmaceuticalcomposition to be employed will depend, for example, upon thetherapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will varydepending, in part, upon the molecule delivered, the indication forwhich the human c-fms antigen binding protein is being used, the routeof administration, and the size (body weight, body surface or organsize) and/or condition (the age and general health) of the patient. Incertain embodiments, the clinician may titer the dosage and modify theroute of administration to obtain the optimal therapeutic effect.

A typical dosage may range from about 1 μg/kg to up to about 30 mg/kg ormore, depending on the factors mentioned above. In specific embodiments,the dosage may range from 10 μg/kg up to about 30 mg/kg, optionally from0.1 mg/kg up to about 30 mg/kg, alternatively from 0.3 mg/kg up to about20 mg/kg. In some applications, the dosage is from 0.5 mg/kg to 20mg/kg. In some instances, an antigen binding protein is dosed at 0.3mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, or 20 mg/kg. The dosageschedule in some treatment regimes is at a dose of 0.3 mg/kg qW, 0.5mg/kg qW, 1 mg/kg qW, 3 mg/kg qW, 10 mg/kg qW, or 20 mg/kg qW.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular human c-fms antigen binding protein in the formulation used.Typically, a clinician administers the composition until a dosage isreached that achieves the desired effect. The composition may thereforebe administered as a single dose, or as two or more doses (which may ormay not contain the same amount of the desired molecule) over time, oras a continuous infusion via an implantation device or catheter.Appropriate dosages may be ascertained through use of appropriatedose-response data. In certain embodiments, the antigen binding proteinscan be administered to patients throughout an extended time period.Chronic administration of an antigen binding protein minimizes theadverse immune or allergic response commonly associated with antigenbinding proteins that are not fully human, for example an antibodyraised against a human antigen in a non-human animal, for example, anon-fully human antibody or non-human antibody produced in a non-humanspecies.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. In certain embodiments, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

The composition also may be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of the desired molecule maybe via diffusion, timed-release bolus, or continuous administration.

It also may be desirable to use human c-fms antigen binding proteinpharmaceutical compositions according to the disclosed ex vivo. In suchinstances, cells, tissues or organs that have been removed from thepatient are exposed to human c-fms antigen binding proteinpharmaceutical compositions after which the cells, tissues and/or organsare subsequently implanted back into the patient.

In particular, human c-fms antigen binding proteins can be delivered byimplanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptide. In certain embodiments, such cells may be animal or humancells, and may be autologous, heterologous, or xenogeneic. In certainembodiments, the cells may be immortalized. In other embodiments, inorder to decrease the chance of an immunological response, the cells maybe encapsulated to avoid infiltration of surrounding tissues. In furtherembodiments, the encapsulation materials are typically biocompatible,semi-permeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the scope of the appended claims.

EXAMPLES Assays

AML-5 Assays

In order to determine whether antibodies directed against c-fms can bindand exhibit functional activity in blocking the c-fms/CSF-1 axis, acell-based bioassay was used. This assay quantitatively measuresCSF-1-driven proliferation of a growth-factor dependent humanmyelomonocytic cell line, AML5 (University Health Network, Toronto,Ontario). The assay therefore, measures the inhibition of thisproliferation by introducing agents that block this pathway. In thisassay, AML-5 cells were incubated with 10 ng/ml CSF-1 in the presence ofdecreasing concentrations of antibody. After 72 hours, cellproliferation was measured using Alamar Blue™ (Biosource), an indirectmeasure of proliferation based on metabolic activity of the cells.

Bone Marrow Assays

In a similar assay to determine whether the antibodies could cross-reactwith cynomolgus monkey c-fms, antibodies were tested in CSF-1-drivenproliferation of the monocytic cells from primary monkey bone marrow.Similar to the AML-5 proliferation assay, cynomolgus bone marrow cellswere incubated with 10 ng/ml CSF-1 in the presence of decreasingconcentrations of antibody. After 72 hours, cell proliferation wasmeasured using Alamar Blue.

Antibody Clones Used in Experiments

The following experiments include the use of three antibody clones,designated as 1.109, 1.2, 2.360, which all are tetramers including twocomplete heavy and two complete light chains. Clone 1.109 comprises twoheavy chains H1 (SEQ ID NO:4) and two light chains L1 (SEQ ID NO:36),clone 1.2 comprises two heavy chains H8 (SEQ ID NO:11) and two lightchains L8 (SEQ ID NO:43), and clone 2.360 comprises two heavy chains H24(SEQ ID NO:27) and two light chains L22 (SEQ ID NO:57).

Example 1 Preparation of c-fms Hybridomas

Embodiments may employ the XenoMouse® technology to develop fully humanmonoclonal antibodies directed against human c-fms. For immunizationpurposes, the c-fms-Fc, a human c-fms extracellular domain (residues1-512, see, FIG. 8; SEQ ID: 1) with a C-terminal human Fc domain wasemployed. In addition, c-fms-LZ, a human c-fms extracellular domain(residues 1-512) with a C-terminal leucine zipper domain (Amgen Lot#45640-43) and 293T/c-fms cell line, a human adenovirus type5-transformed human embryonic kidney cell line transfected withfull-length human c-fms were utilized to screen the anti-c-fmsantibodies.

Cohort 1 (IgG₁) and cohort 2 (IgG₂) XenoMice® were immunized/boostedwith c-fms-Fc. Serum titers were measured by enzyme-linkedimmunoabsorbent assay (ELISA) and spleens from both cohorts 1 and 2 micewere fused to generate hybridomas. The resulting polyclonal supernatantswere screened for binding to c-fms-LZ by ELISA and 293T/c-fms cells byfluorometric microvolume assay technology (FMAT). A total of 828positive supernatants were tested for inhibition of CSF-1 binding to thec-fms/293T cells by fluorescence-activated cell sorting (FACS). Theresulting 168 positive supernatants were further tested for inhibitionof CSF-1-induced proliferation of acute myelogenous leukemia (AML)-5cells. Based on the screening, 33 hybridomas were identified asantagonistic to CSF-1 activity and were selected for cloning.

Example 2 Characterization of Anti-c-fms Hybridomas

From the 33 selected hybridomas, 29 (19 IgG1 and 10 IgG2 isotypes) weresuccessfully cloned and supernatants from these clones were tested forinhibition of CSF-1 binding to the 293T/c-fms cells and inhibition ofCSF-1 induced proliferation of AML-5 cells. A low-resolution Biacorebinding assay using monomeric c-fms protein indicated that the K_(D) ofthese 29 anti-c-fms hybridomas was in the range of 0.1-43 nM (see TABLE8). Anti-human IgG was immobilized on all four flow cells of a sensorchip using amine coupling. Crude hybridoma samples were diluted intohalves and captured on the anti-IgG surface. Monomeric c-fms (residues1-512)-pHis was the analyte at a concentration of 125 nM. Sequencelineage analyses were also performed on the 29 hybridomas (see, FIG. 2).

TABLE 8 Low Resolution Biacore Binding Results For Anti-C-fms HybridomasmAb mAb Clone Clone k_(a) (1/Ms) k_(d) (1/s) K_(D) (nM) 1.109.1 13.85E+05 4.36E−05 0.1 2.131.2 2 4.30E+04 1.00E−05 0.2 2.508.1 3 7.49E+045.28E−05 0.7 1.33.1.1 4 9.89E+04 1.16E−04 1.2 1.2.1 5 8.17E+05 1.13E−031.4 1.42.3 6 6.60E+05 1.07E−03 1.6 1.64.1 7 2.55E+05 6.40E−04 2.5 1.30.28 4.06E+05 1.40E−03 3.4 1.134.1 9 1.53E+05 7.03E−04 4.6 2.475.2 103.00E+05 1.40E−03 4.7 2.103.1 11 7.51E+04 3.64E−04 4.8 1.39.3 121.13E+05 6.08E−04 5.4 1.72.2 13 1.03E+05 5.68E−04 5.5 2.360.3 146.05E+04 3.38E−04 5.6 1.13.2 15 1.20E+05 7.73E−04 6.4 2.65.2 16 1.68E+051.60E−03 9.5 1.143.2 17 5.50E+04 5.59E−04 10 1.90.2 18 1.93E+05 2.00E−0310 1.144.1 19 2.30E+05 2.50E−03 11 1.26.1 20 2.39E+05 2.63E−03 112.369.3 21 1.00E+05 1.38E−03 14 1.16.2 22 1.29E+05 2.96E−03 23 1.66.1 232.39E+05 5.50E−03 23 2.550.1 24 3.00E+05 7.24E−03 24 2.291.2 25 2.86E+059.30E−03 33 1.27.3 26 2.99E+05 1.00E−02 33 1.34.3 27 3.65E+05 1.31E−0236 1.131.1 28 5.31E+04 2.15E−03 41 2.534.1 29 3.45E+05 1.50E−02 43

Based on the binding inhibition and proliferation inhibition assays, 16of the 29 supernatants (eleven IgG1 and five IgG2 isotypes) wereselected for further characterization. Cross-reactivity to mouse andcynomolgus c-fms was tested by inhibition of CSF-1-induced proliferationof mouse DRM (a ras- and myc-immortalized monocytic cell line derivedfrom Dexter type culture of mouse bone marrow) cells and primarycynomolgus bone marrow cells, respectively. With respect to cellproliferation, none of the supernatants inhibited the proliferation ofthe mouse DRM cells (data not shown) while 13 of 16 supernatantsinhibited proliferation of the cynomolgus bone marrow cells. Thesupernatants were also tested for inhibition of CSF-1-inducedproliferation of human peripheral blood-derived CD14⁺ monocytes andretested in the human AML-5 bioassay (see Assay section above), theresults of which are shown in TABLES 9 and 10. The 2-4A5 antibody(Biosource), which is a rat anti-human c-fms antibody, was used as apositive control.

Four IgG₁ isotype antibodies had <10 pM potency in the AML-5 bioassayand three of the antibodies, Clone ID Nos. 1.2.1, 1.109.3, and 1.134.1,inhibited the proliferation of cynomolgus bone marrow cells. Of thethree antibodies, clones 1.2.1 and 1.109.3 had the highest affinity forc-fms in the Biacore binding assay. Two IgG₂ isotype antibodies, 2.103.3and 2.360.2, showed high potency in the AML-5 and cynomolgus bone marrowbioassays and similar affinities for c-fms in the Biacore assay. Thefive antibodies that showed high potency in the AML-5 and cynomolgusbone marrow bioassays also showed diversity in sequence. Based on thesefactors of potency, affinity, and diversity, clones 1.2.1, 1.109.3, and2.360.2 were chosen for additional development and characterization.

TABLE 9 Summary Of Bioassay Results For The Anti-C-fms HybridomaSupernatants Cynomolgus Human CD14+ AML-5 bone marrow monocytes bioassaybioassay bioassay (mean, n = Clone (mean, n = 2) (n = 1) 3 or 4) ID IC₅₀(pM) IC₅₀ (pM) IC₅₀ (pM) 1.2.1 40 <7 6 1.26.1 33 13 27 1.27.2 933 200 731.30.3 267 67 40 1.39.2 67 67 27 1.42.3 200 67 20 1.64.2 NA* 13 4 1.66.247 67 27 1.109.3 73 20 5 1.134.1 40 <7 7 1.143.1 360 ND** 100 2.103.3 27133 53 2.360.2 53 67 27 2.475.2 167 67 40 2.508.2 NA* 133 20 2.534.2 NA*47 20 2-4A5 8333 667 187 c-fms-Fc 617 556 210 Anti- 3333 667 20 CSF-1*NA = No Activity, Not Cross-Reactive; **ND = Not Done

TABLE 10 Synopsis Of Bioassay Results For Anti-c-fms HybridomaSupernatants Concentration of Half-Max (IC₅₀, ng/ml) Cynomolgus HumanCD14+ AML-5 bone marrow monocytes bioassay bioassay bioassay (mean, n =AML-5 × Clone (mean, n = 2) (n = 1) 3 or 4) Difference 1.2 6 <1 0.9 41.26.1 5 2 4 24 1.27.2 140 30 11 44 1.30.3 10 10 6 24 1.39.2 30 10 4 241.42.3 10 3 9 1.64.2 NA* 2 0.6 8 1.66.2 7 10 4 12 1.109.3 11 3 0.7 1.41.134.1 6 <1 1 3 1.143.1 54 ND** 15 119 2.103.1 4 20 8 19 2.360.2 8 10 411 2.475.2 25 10 6 18 2.508.2 NA* 20 5 20 2.534.2 NA* 7 3 12 2-4A5 1250100 28 c-fms-Fc 100 90 34 Anti- 500 100 3 (n = 1) CSF-1 *NA = NoActivity, not cross-reactive; **ND = Not Done.

Example 3 Expression and Characterization of Antibodies

Heavy and light chain genes for antibody clones 1.2, 1.109, and 2.360were isolated and cloned into constructs for expression as IgG₂ heavychains and kappa light chains. Antibodies were expressed by transientexpression in COS/PKB cells and purified by Protein A chromatography.Antibody yields were 3.6-7.4 mg/l, which is within the expected rangefor this expression system.

The activities of the cloned antibodies and the hybridoma-expressedantibodies were compared in the AML-5 proliferation assay. AML-5 cellswere incubated with 10 ng/ml CSF-1 in the presence of decreasingconcentrations of antibody. After 72 hours, cell proliferation wasmeasured using Alamar Blue (see, FIG. 3). The recombinant antibodiesshowed similar neutralizing activity as the hybridoma supernatants, andconversion of 1.2 and 1.109 from IgG1 to IgG2 had no apparent effect.The recombinant antibodies also demonstrated good neutralizing activityin the cynomolgus proliferation assay as shown in FIG. 4. Similar to theAML-5 proliferation assay, cynomolgus bone marrow cells were incubatedwith 10 ng/ml CSF-1 in the presence of decreasing concentrations ofantibody. After 72 hours, cell proliferation was measured using AlamarBlue.

Characterization of the purified antibodies by SDS-PAGE andsize-exclusion chromatography (SEC) produced typical results, with theexception of the clone 1.109 light chain, which migrated larger thanexpected on SDS-PAGE. This exception was not unexpected because anN-linked glycosylation site sequence was previously noted in CDR1. Thelarger than expected migration suggested that this glycosylation sitewas occupied.

N-terminal sequencing of the antibodies confirmed that signal peptideswere processed as expected and that the heavy chain N-terminal glutamineresidues were likely cyclized to pyroglutamic acid as would be expected.Mass spectrometry was performed on the individual antibody chainsfollowing enzymatic deglycosylation. The masses of the heavy chainsconfirmed that the N-terminal glutamine residues were cyclized topyroglutamic acid and that the C-terminal lysine residues were absent.No other post-translational modifications were noted. The masses of theclone 1.2 SM and 2.360 light chains confirmed that they were intact withno post-translational modifications. A mass was not obtained for theclone 1.109 light chain, probably because the glycosylation site wasresistant to enzymatic removal and thus an accurate mass could not beobtained.

Example 4 Correction of Somatic Mutations (SMs)

Sequence comparison of IgG2 clone 1.2, 1.109 and 2.360 antibodies toknown germline sequences revealed the following somatic mutations, asshown in TABLE 11, with the numbering in the table being with respect tothe mature sequence as shown in FIGS. 1A and 1B.

TABLE 11 Somatic Mutations Relative To The Closest Germline SequenceAntibody Germline Chain Somatic Mutation Residue Comments 1.2 LC Ser-78in FR3 Thr 1.2 HC None 1.109 LC Asn-28 in CDR1; Asp: Lys Asn-28 createsan Asn-45 in FR2 N-linked glycosylation site 1.109 HC None 2.360 LCGln-45 in FR2 Lys 2.360 HC Val-79 in FR3 Ala

To test if the somatic mutations could be converted to germlineresidues, the relevant constructs were generated and antibodies wereexpressed by transient expression in COS/PKB cells and purified byProtein A chromatography. These antibodies were designated IgG₂ clone1.2 SM, 1.109 SM, and 2.360 SM (SM=somatic mutation cured). For the1.109 LC, two constructs were made. In the first construct, Asn-28 wasconverted to Asp-28 to eliminate the N-linked glycosylation site, and inthe second construct, Asn-28 was converted to Asp-28 and Asn-45 wasconverted to Lys-45. Yields were 1.7-4.5 mg/l, which is within theexpected range for this expression system.

Characterization of the purified antibodies by SDS-PAGE andsize-exclusion chromatography (SEC) produced typical results. SDS-PAGEof the two forms of IgG₂ clone 1.109 SM showed that the light chainmigrated faster than the parent antibody light chain, confirming thatthe N-linked glycosylation site was eliminated. N-terminal sequencingshowed that the N-termini of the antibody chains were intact, and massspectrometry showed that the somatic mutations had been converted togermline residues.

Example 5 Characterization of Somatic Mutation-Corrected Antibodies

Following the correction of the somatic mutations, the purified IgG₂antibodies were retested in the AML-5 and cynomolgus bone marrowproliferation assays. The IC₅₀ in the AML-5 proliferation assay did notchange for IgG₂ clone 1.2 SM or 1.109 SM (SM=somatic mutation cured)relative to the parent IgG₂ antibodies, but there was a 10-fold loss inpotency for the IgG₂ clone 2.360 SM antibody (see, TABLE 12).

The binding affinities of the somatic mutation corrected antibodies tomonomeric c-fms protein were measured by surface plasmon resonance usinga Biacore 3000 instrument. The affinity of IgG₂ clone 1.2 SM antibodywas essentially unchanged from the parent antibodies, whereas theaffinities of the IgG₂ clone 1.109 SM and 2.360 SM antibodies were˜2-fold less than the respective parent antibodies (see, TABLE 12).

The parent (PT) and SM IgG₂ antibodies were further tested for theability to inhibit binding of ¹²⁵I-hCSF-1 to AML-5 cells. The apparentbinding affinity of ¹²⁵I-hCSF-1 to AML-5 cells was first determined tobe 46 pM and the K_(I) of unlabeled hCSF-1 was 17.8 pM (see, Example10). As shown in Table 12, the K_(I) value for antibody 1.2 was in linewith the IC₅₀ value in the AML-5 bioassay and 1.2 SM gave similarresults. The K_(I) value for antibody 1.109 was also in line with theIC₅₀ value and there was no change with the 1.109 SM antibody despite a2-fold loss in affinity for monomeric c-fms as measured by Biacore.Antibody 2.360 did not inhibit as well as antibodies 1.2 and 1.109, and2.360 SM inhibited less well than the parent antibody.

TABLE 12 Properties Of The Parent (PT) Versus Germlined Antibodies (SM)Inhibition Binding Cynomolgus (K_(I)) Affinity (K_(D)) AML-5 bone marrowof ¹²⁵I-CSF-1 to monomeric bioassay bioassay binding to c-fms byAntibody IC₅₀ (pM) IC₅₀ (pM) AML-5 (pM) Biacore (pM) 1.2 27 78 8.5 5161.2 SM* 12 81 11.5 548 1.109 27 16 13.5 51 1.109 SM* 23 9.7 102 2.360 6067 ~160 535 2.360 SM* ~900 1200 *SM = somatic mutation cured

The activity of the 1.2 SM antibody was further investigated inproliferation assays using human or Cynomologous bone marrow monocyticcells. For the human assay, human cells were incubated with 11.1 ng/mlrecombinant human CSF-1 in the presence of decreasing concentrations ofantibody 1.2 SM. For the cynomolgus assay, cynomolgus cells wereincubated with 29.63 ng/ml recombinant human CSF-1 in the presence ofdecreasing concentrations of antibody 1.2 SM. Human IgG2 antibody wasused in control experiments. After 7 days, cell proliferation wasmeasured using CellTiter-Glo (Promega, Madison Wis.) to determine levelsof ATP. Nonlinear regression curve fit was performed to determine theIC50 of the antibody. TABLE 13 shows the results of three sets ofexperiments.

TABLE 13 Activity of clone 1.2 SM antibody in cell proliferation assaysBone marrow IC50 of 1.2 SM in the Presence of hCSF-1 (pM) monocytic cellExpt. 1 Expt. 2 Expt. 3 Average S.D. Human 15.5 20.1 10.9 15.5 4.6Cynomolgus 42.55 26.01 22.90 32.73 13.89

Example 6 Inhibition of c-fms Tyrosine Phosphorylation

To show that anti-c-fms IgG₂ mAbs, 1.109, 1.2 and 2.360, are capable ofcomplete or nearly complete inhibition of phosphotyrosine(pTyr)-response, 293T/c-fms cells were treated with these mAbs for 1hour at various concentrations at 37° C. prior to CSF-1 stimulation.

Various concentrations of the IgG₂ mAbs using titration dilutions wereat 1.0, 0.1, 0.01, 0.001 and 0.0001 μg/ml. As controls, a non-blockinganti-c-fms monoclonal antibody, mAb 3-4A4 (BioSource, Intl.) and anon-relevant antibody, hCD39 M105, each at 1.0 μg/ml, were used.Serum-starved 293T/c-fms cells were treated with each of the IgG₂ (PT)mAbs using the various concentrations as mentioned above, and eachconcentration varying in a ten-fold dilution prior to a five-minuteCSF-1 stimulation at 50 ng/ml for 5 minutes. Following stimulation,whole-cell lysates were collected, immunoprecipitated at 4° C. overnightwith an anti-c-fms C20 polyclonal antibody (Santa Cruz Biotechnology,Inc.) and examined by Western blotting wherein the blot was immunoprobedwith a generic anti-pTyr antibody, 4G10 (Upstate Biotechnology), and ananti-c-fms C20 antibody for the levels of tyrosine phosphorylation ofc-fms and c-fms itself, respectively.

To grow the 293T/c-fms cells on 24×10 cm dishes, at 37° C. in 5% CO₂,eleven T175 flasks (˜50-60% confluent) were collected via 4 mltrypsin/flask (Gibco-Invitrogen) and transferred to 70 ml DMEM(Gibco)/10% FBS (JRH Biosciences). Each 10 cm dish was then given 10 mlmedium and inoculated with 2 ml of the collected cells. DMEM/-FBS mediumwas prepared for 1 hour at 37° C. Culture medium from 10 cm dishes wasremoved via careful aspiration, removing as much FBS-containing mediumas possible. Ten ml DMEM/-FBS was added and the mixture was incubatedfor 1 hour at 37° C.

After serum starvation for 1 hr at 37° C., the medium was removed.Antibody treatments and minus-Ab controls were set up with either 4.0 mlof the serially-diluted Ab-containing samples or DMEM/-FBS alone, andfurther incubated for another 1 hour at 37° C. to provide a total of 2hours of serum starvation. The antibody pre-treatment and ligandstimulation is illustrated in TABLE 14.

TABLE 14 Dish Ab Pre-Treatment Ligand Stimulation #1 DMEM/−FBS (minusAb) medium alone #2 DMEM/−FBS (minus Ab) CSF-1 #3 CLONE1.109 @ 1.0 μg/mlmedium alone #4 CLONE 1.2 @ 1.0 μg/ml medium alone #5 CLONE 2.360 @1.0μg/ml medium alone #6 3-4A4 @ 1.0 μg/ml medium alone #7 M105 @1.0 μg/mlmedium alone #8 CLONE 1.109 @ 1.0 μg/ml CSF-1 #9 CLONE 1.109 @ 0.1 μg/mlCSF-1 #10 CLONE 1.109 @ 0.01 μg/ml CSF-1 #11 CLONE 1.109 @ 0.001 μg/mlCSF-1 #12 CLONE 1.109 @ 0.001 μg/ml CSF-1 #13 CLONE 1.2 @ 1.0 μg/mlCSF-1 #14 CLONE1.2 @ 0.1 μg/ml CSF-1 #15 CLONE1.2 @ 0.01 μg/ml CSF-1 #16CLONE1.2 @ 0.001 μg/ml CSF-1 #17 CLONE1.2 @ 0.0001 μg/ml CSF-1 #18CLONE2.360 @ 1.0 μg/ml CSF-1 #19 CLONE2.360 @ 0.1 μg/ml CSF-1 #20CLONE2.360 @ 0.01 μg/ml CSF-1 #21 CLONE2.360 @ 0.001 μg/ml CSF-1 #22CLONE2.360 @ 0.0001 μg/ml CSF-1 #23 3-4A4 @ 1.0 μg/ml CSF-1 #24 M105 @1.0 μg/ml CSF-1

A fresh vial of CSF-1 (R&D Systems/216-MC/Lot CC093091) at 50 ng/μl (25μg/vial) was reconstituted in 500 μl PBS (Gibco)/0.1% BSA (Sigma) andkept on ice. A 1:1000 dilution of CSF-1 stock (60 μl) was prepared intoDMEM/-FBS (60 ml) approximately 5 minutes prior to the end ofAb-treatment. The 293T/c-fms cells were incubated with 50 ng/ml of CSF-1for 5 minutes at 37° C. The supernatants were removed and 2 ml of coldlysis buffer (100 ml PBS/1% Triton, 100 μl 0.5 M EDTA, 100 μl 1.0 M NaF,200 μl 0.5 M beta-glycerol phosphate, 500 μL sodium vanadate (100×),10.0 μL okadaic acid (10,000×), and 4 tablets of Complete ProteaseInhibitor) was added. The lysates (2.0 ml) were combined with 30 μl 50%Protein A/G Sepharose (Amersham) and incubated for 1 hour at 4° C. on arocker platform to pre-clear non-specific binding proteins. Afterspinning the fractions, the supernatants were decanted onto fresh 15 mltubes.

Whole cell lysates were immunoprecipitated with 2.5 μg/ml of anti-c-fmsC20 (25 μl; at 0.2 μg/μl). The antibody-cell lysate mixtures wereincubated overnight at 4° C. on the rocker platform to probe for totalc-fms. Donkey anti-rabbit IgG/HRP (1:10,000 in blocking solution;Jackson) was added and further incubated for another 30 min at 4° C. Theimmunocomplexes were run on SDS-PAGE and immunoblotted. The Westernblots were probed with either anti-pTyr 4G10 or anti-c-fms C20 for thedetection of pTyr c-fms and total c-fms, respectively.

As shown in FIG. 5, IgG₂ clones (PT) 1.109, 1.2 and 2.360 exhibited theability to inhibit ligand-induced pTyr/c-fms in the 293T3/c-fms assaysystem. Treatment with 0.1 mg/ml (8.3 nM) of IgG₂ clone (PT) 1.109 or1.2 for 1 hour prior to CSF-1 stimulation reduced the phosphotyrosinesignal to background levels. On the other hand, IgG₂ clone (PT) 2.360produced equal inhibition at 1.0 μg/ml (83 nM). However, treatment ofeither antibodies at 0.01 mg/ml (0.83 nM) or less did not result in pTyrinhibition. In contrast, non-blocking anti-c-fms 3-4A4 and anon-relevant hCD39 M105 antibody, at the highest dose (1.0 mg/ml) had noeffect on ligand-induced pTyr signal compared to the -mAb/+CSF-1control. Thus, the inhibition of pTyr formation is directly linked tothe blocking of CSF-1 binding.

Assuming that the 293T/c-fms transfectants used in these assays retainedthe previously measured cell-surface c-fms density of ˜30,000receptors/cell, ˜3 million cells would bear ˜90×10⁶ c-fms, significantlyless than the ˜5.0×10¹¹ mAb in 4.0 ml pretreatment at 0.1 mg/ml. mAbpresent at ˜10,000 fold excess with respect to target makes saturationof available c-fms likely. This indicates that 8.3 nM clone (PT) 1.109or 1.2 effectively blocked signaling of CSF-1 at 50 ng/ml or 1.0 nM, oran approximate 10:1 (mAb:c-fms) molar ratio. The threshold ofeffectiveness for clones (PT) 1.109 and 1.2 likely falls between 0.1 and0.01 mg/ml (0.83-8.3 nM) in this assay system.

Treatment with 1.0 μg/ml IgG₂ (PT) mAbs in the absence of CSF-1 additiongave no pTyr signal above background levels. Previous experiments withall three IgG₂ (PT) forms used at 10 mg/ml also revealed no pTyr signalunder the same conditions. There was no measured agonistic activityassociated with these mAbs.

Example 7 Inhibition of Ligand-Induced pTyr/c-fms Using IgG₂PT and SMForms

The purpose of this study was to determine if there is any functionalchanges in the germline-reverted (SM) forms of IgG₂ clones 1.109, 1.2and 2.360, as compared with their respective parent forms (PT). Toprepare the 293T/c-fms cells for this experiment, cells growing fromfive T175 (˜80-90% confluent) were collected via 4 ml trypsin/flask andtransferred to 75 ml DMEM with 10% FBS. To each 24×10 cm dish, 9 mlmedium was added and inoculated with 3 ml of the collected cells.DMEM/-FBS medium was prepared, and/or warmed for 1 hour at 37° C.Culture medium was removed from 10 cm dishes via careful aspiration toremove as much FBS-containing medium as possible. DMEM/-FBS (10 ml) wasadded and the mixture was incubated for 1 hour at 37° C. Cold lysisbuffer was prepared and kept on ice.

Monoclonal antibody titrations, as depicted in TABLE 15, were preparedand kept at room temperature.

TABLE 15 Titration of IgG₂ C-fms Monoclonal Antibodies mAb (μg/μL)Volume Used DMEM/−FBS Clone 1.109 PT (0.41) 14.6 μL 6.0 ml Clone 1.109SM (G) (0.34) 17.6 μL 6.0 ml Clone 1.109 SM (F/G)(0.58) 10.3 μL 6.0 mlClone 1.2 PT (1.57) 3.8 μL 6.0 ml Clone 1.2 SM (0.35) 17.1 μL 6.0 mlClone 2.360 PT (0.41) 14.6 μL 6.0 ml Clone 2.360 SM (0.55) 10.9 μL 6.0ml 3-4A4 (0.2) 15 μL 3.0 ml

A series of serial antibody dilutions (300 μL+2.7 ml DMEM/-FBS) weretested within the range of 1.0 μg/ml to 0.1 μg/ml for each mAb. After 1hour of serum starvation at 37° C., the medium was removed and antibodypre-treatments and minus-Ab controls were prepared similarly asdescribed in Table 13. The antibody pretreatments and ligand stimulationis described in TABLE 16 hereinbelow:

TABLE 16 Dish No. Ab-pretreatment Ligand Stimulation 1 DMEM/−FBS (minusAb) medium alone 2 DMEM/−FBS (minus Ab) CSF-1 3 3-4A4 @ 1.0 μg/ml CSF-14 Clone 1.109 @ 1.0 μg/ml medium alone 5 Clone 1.109 @ 1.0 μg/ml CSF-1 6Clone 1.109 @ 0.1 μg/ml CSF-1 7 Clone 1.109 SM-G @ 1.0 μg/ml mediumalone 8 Clone 1.109 SM-G @ 1.0 μg/ml CSF-1 9 Clone 1.109 SM-G @ 0.1μg/ml CSF-1 10 Clone 1.109 SM-F/G @ 1.0 μg/ml medium alone 11 Clone1.109 SM-F/G @ 1.0 μg/ml CSF-1 12 Clone 1.109 SM-F/G @ 0.1 μg/ml CSF-113 Clone 1.2 @ 1.0 μg/ml medium alone 14 Clone 1.2 @ 1.0 μg/ml CSF-1 15Clone 1.2 @ 0.1 μg/ml CSF-1 16 Clone 1.2 SM @ 1.0 μg/ml medium alone 17Clone 1.2 SM @ 1.0 μg/ml CSF-1 18 Clone 1.2 SM @ 0.1 μg/ml CSF-1 19Clone 2.360 @ 1.0 μg/ml medium alone 20 Clone 2.360 @ 1.0 μg/ml CSF-1 21Clone 2.360 @ 0.1 μg/ml CSF-1 22 Clone 2.360 SM @ 1.0 μg/ml medium alone23 Clone 2.360 SM @ 1.0 μg/ml CSF-1 24 Clone 2.360 SM @ 0.1 μg/ml CSF-1

Ligand-induced pTyr by anti-c-fms mAbs (SM forms) was performed asdescribed in Example 6 for the PT forms.

Experiments using the 293T/c-fms cells to compare the effects of PTversus SM forms of the three IgG₂ mAbs at 1.0 and 0.1 μg/ml revealed nodifferences in the ability to inhibit ligand-induced pTyr/C-fms (seeFIG. 6). Clones 1.109 and 1.2 (both PT and SM forms) showed inhibitionat lower concentrations than with the 2.360 (PT and SM forms).

Clones 1.109 and 1.2 (PT or SM) were able to prevent ligand-inducedpTyr/c-fms in vitro when 293T/c-fms cells were treated with 0.1 μg/ml(8.3 nM) or greater mAb for 1 hour at 37° C. prior to the addition ofCSF-1 at 50 ng/ml (1.0 nM). The ability of these monoclonal antibodiesto block the formation of pTyr/c-fms would lead to the inhibition ofCSF-1 signaling, monocyte migration and, subsequently, accumulation ofTAMs. No agonistic activity appeared to be associated with these mAbs,to avoid activating the receptor in a non-CSF-1 dependent manner. ThemAbs showed no agonistic activity when used at a concentration of 1.0μg/ml and as high as 10 μg/ml (data not shown).

Accordingly, the mAbs were able to prevent ligand-induced pTyr/c-fms invitro.

Example 8 Immunoprecipitation of c-fms by Anti-c-fms mAbs

The ability of the IgG₂ anti-c-fms mAbs to bind and immunoprecipitatec-fms was achieved by using the stably-transfected 293T/c-fms cells asdescribed above. Whole-cell lysates of unstimulated cells wereimmunoprecipitated overnight with each mAb (PT and SM) and anti-c-fmsC20 antibody and examined via Western blot with C20 Ab (Santa CruzBiotechnology, Inc.) as the probe for detection of c-fms. C-fms wasimmunoprecipitated by monoclonal antibodies. Lysates of stablytransfected 293T/c-fms grown at 37° C./5% CO₂ to ˜75% confluency wereprepared and combined with the monoclonal antibodies, as shown in TABLE17.

TABLE 17 Tube No. Ab (μg/μL) Ab (μL) 1 Clone 1.109 (0.41) 6.1 2 Clone1.109 SM F/G (0.58) 4.3 3 Clone 1.2 (1.57) 1.6 4 Clone 1.2 SM (0.35) 7.15 Clone 2.360 (0.41) 6.1 6 Clone 2.360 SM (0.52) 4.8 7 C20 (0.2) 12.5 8C19 (0.2) 12.5

Immunoprecipitation experiments using untreated whole-cell lysates ofstable 293T/c-fms demonstrated comparable ability of the various mAbs(except 2.360 SM) to bind and precipitate c-fms, in comparison to thepolyclonal anti-c-fms C20 control; clone 2.360-SM, however, exhibited areduced capacity in this assay (see FIG. 7).

Example 9 Immunoprecipitation of SNP-Variants by IgG₂ mAbs

Single nucleotide polymorphisms or SNPs are DNA sequence variations thatoccur when a single nucleotide (A, G, T, or C) in the genomic sequencehas been changed. SNPs may occur in both the coding and non-codingregions of the human genome. Many SNPs have no impact on cell function,but scientists consider other SNPs could predispose people to disease orhave an effect in their drug response. Variations in DNA sequence canhave a major influence on how a person responds to a disease,environmental insults (e.g., bacteria, viruses, toxins, and chemicals),drugs and other therapies. For this reason, SNPs are of great value tobiomedical research, pharmaceutical product development and medicaldiagnosis. Furthermore, SNP maps will enable the scientist in theidentification of multiple genes that are associated with complexdiseases such as cancer, diabetes, and vascular diseases.

The extracellular region of human c-fms can be divided into fiveimmunoglobulin (Ig)-like repeated domains (designated A through E). See,for example, Hampe, A. et al. (1989) Oncogene Res. 4:9-17 for adiscussion of the human domains. See, for example, Wang, et al. (1993)Molecular and Cell Biology 13:5348-5359 for the corresponding domains inthe mouse protein. Domains A-C had been shown to comprise the CSF-1binding region, while domain D had been shown to help regulate receptordimerization upon binding of ligand. Three naturally-occurringSNP-variants of human c-fms were prepared, namely, A245S, V279M inIg-Domain C and H362R in Ig-Domain D (see FIG. 8 for amino acid sequenceof the extracellular domain of c-fms). These SNPs are found either in ornear the CSF-1 binding region and examination by Western blots probedwith anti-c-fms H-300 (a rabbit polyclonal antibody raised against aminoacids 11-310 mapping near the N-terminus of human c-fms/CSF-1R; SantaCruz Biotech., Inc., Cat. No. sc-13949).

To study how human c-fms SNP variants interact with the various c-fmsAbs provided herein, transiently-transfected 293T cells expressing thethree types of c-fms SNP variants, as discussed above, and wildtype (WT)c-fms (as well as an irrelevant, vector-matched control) were used toassess the ability of each anti-c-fms mAb to bind SNP-variants viaimmunoprecipitation.

293T cells were transfected in duplicate 10-cm dishes with c-fms A245S,V279M, H362R, WT c-fms and an irrelevant control construct in themammalian expression vector pCIneo and grown for 48 hours at 37° C./5%CO₂. Cell lysates were prepared as described above. Anti-c-fms mAbs andpolyclonal anti-c-fms C20 or anti-c-kit C19 at 2.5 μg/ml in 1.0 mlaliquots were added to each lysate as illustrated in TABLE 18.

TABLE 18 Tube Transfectant IP Ab (μg/μL) IP Ab (μL) 1 c-fms Clone 1.2(1.57) 1.6 2 c-fms Clone 1.2 SM (0.5) 5.0 3 c-fms Clone 1.109 (0.41) 6.14 c-fms Clone 1.109 SM (0.5) 5.0 5 c-fms Clone 2.360 (0.41) 6.1 6 c-fmsClone 2.360 SM (0.5) 5.0 7 c-fms C20 (0.2) 12.5 8 c-fms C19 (0.2) 12.5 9A245S Clone 1.2 (1.57) 1.6 10 A245S Clone 1.2 SM (0.5) 5.0 11 A245SClone 1.109 (0.41) 6.1 12 A245S Clone 1.109 SM (0.5) 5.0 13 A245S Clone2.360 (0.41) 6.0 14 A245S Clone 2.360 SM (0.5) 5.0 15 A245S C20 (0.2)12.5 16 V279M Clone 1.2 (1.57) 1.6 17 V279M Clone 1.2 SM (0.5) 5.0 18V279M Clone 1.109 (0.41) 6.1 19 V279M Clone 1.109 SM (0.5) 5.0 20 V279MClone 2.360 (0.41) 6.1 21 V279M Clone 2.360 SM (0.5) 5.0 22 V279M C20(0.2) 12.5 23 H362R Clone 1.2 (1.57) 1.6 24 H362R Clone 1.2 SM (0.5) 5.025 H362R Clone 1.109 (0.41) 6.1 26 H362R Clone 1.109 SM (0.5) 5.0 27H362R Clone 2.360 (0.41) 6.1 28 H362R Clone 2.360 SM (0.5) 5.0 29 H362RC20 (0.2) 12.5 30 Minus control Clone 1.2 (1.57) 1.6 31 Minus controlClone 1.2 SM (0.5) 5.0 32 Minus control Clone 1.109 (0.41) 6.1 33 Minuscontrol Clone 1.109 SM (0.5) 5.0 34 Minus control Clone 2.360 (0.41) 6.135 Minus control Clone 2.360 SM (0.5) 5.0 36 Minus control C20 (0.2)12.5 37 Minus control C19 (0.2) 12.5

The cells were incubated overnight at 4° C. on a rocker as described inExample 6.

The antibodies revealed no loss of ability to bind the SNP formscompared to WT control (FIG. 9). The mAbs appear to have the capabilityof binding to the range of naturally occurring c-fms variants.

Immunoprecipitation from untreated whole-cell lysates of stable293T/c-fms demonstrated equal ability of all of the various mAbs (except2.360 SM) to bind and precipitate c-fms compared to polyclonalanti-c-fms control; clone 2.360 SM exhibited a clearly reduced capacityin this assay. Examination of the ability of the various mAbs toimmunoprecipitate c-fms and SNP variants from transiently-transfected293T/c-fms cells revealed no loss of ability to bind the SNP forms. Theability of the various mAbs to bind the c-fms SNPs indicated that theyrecognize c-fms proteins across the spectrum of variants known to existfor humans.

Example 10 Inhibition of ¹²⁵I-hCSF-1 Binding By Anti-c-fms mAbs

The affinity of anti-c-fms mAbs to cell surface expressed human c-fmswas determined by measuring inhibition of ¹²⁵I-hCSF-1 binding to AML-5cells.

Recombinant hCSF-1 (Amgen) was iodinated using ¹²⁵I (Amersham) andIODO-GEN® (Pierce). Seventy-five μl PBS, 10 μg hCSF-1, and 1 mCi ¹²⁵Iwere added to an IODO-GEN® pre-coated iodination tube and left on icefor 15 minutes. The mixture was transferred to an equilibrated 2 ml P6column where ¹²⁵I-hCSF-1 was separated from free ¹²⁵I by gel filtration.Fractions containing iodinated hCSF-1 were pooled, then diluted to aconcentration of 100 nM in binding media (RPMI-1640 with 2.5% bovinealbumin Fraction V, 20 mM Hepes, and 0.2% sodium azide, pH 7.2). Aspecific activity of 4.8×10¹⁵ cpm/mmol was calculated based on theinitial protein concentration of hCSF-1 and a recovery of 80% from acontrol experiment in which an aliquot of ¹²⁵I-hCSF-1 was put throughthe iodination protocol with omission of additional ¹²⁵I.

A saturation radioligand binding experiment was performed in conjunctionwith each inhibition assay in order to determine both a K_(D) and K_(I)for hCSF-1 binding to c-fms expressed on the surface of AML-5 cells.Mixtures were set up in 96-well round-bottom microtiter plates withtotal volumes of 150 μl/well. All reagents were diluted in binding mediacontaining 0.2% sodium azide and experiments were conducted at 4° C. tominimize potential receptor internalization and shedding.

For the saturation binding assay, ¹²⁵I-hCSF-1 was serially diluted2-fold, starting at a concentration of ˜1.7 nM and going out 12 wells toa concentration of ˜1.5 pM. Nonspecific binding was measured at a singleconcentration of ¹²⁵I-hCSF-1 (˜80 pM, in triplicate) in the presence ofa 1,000-fold molar excess of unlabeled hCSF-1, and assumed to be alinear function of the concentration of radiolabeled hCSF-1 present.

For the ¹²⁵I-hCSF-1 inhibition assay, unlabeled hCSF-1 was set up at astarting concentration of 5 nM. Starting concentrations for anti-c-fms1.2, 1.109, and 2.360 (PT and SM for each) were 0.312 nM, 1.25 nM, and20 nM, respectively. Each sample was serially diluted 2-fold out 15wells. Triplicate wells of binding media alone and triplicate wells of1,000-fold molar excess unlabeled hCSF-1 were set up at the beginning,middle, and end of the assay as controls to determine percentinhibition. A single concentration of ¹²⁵I-hCSF-1 (˜9 pM) was added toeach well.

AML-5 cells were washed twice with PBS, and added to each assay plate at1×10⁵ cells/well just prior to incubation.

Both assays were incubated at 4° C. on a miniorbital shaker for 4 hours,the length of time needed to reach equilibrium as determined in timecourse experiments. Two 60 μl aliquots of each incubation mixture weretransferred to chilled 400 μl polyethylene centrifuge tubes containing200 μl phthalate oil and spun for 1.5 minutes in a 4° C. tabletopmicrofuge (Sorvall) at 9615×g to separate cell associated ¹²⁵I-hCSF-1from free ¹²⁵I-hCSF-1. The oil tubes were cut, and each cell pellet andsupernatant collected in individual 12×75 mm glass tubes and loaded on aCOBRA gamma counter (Packard Instrument Company) for cpm measurements.Cpm from duplicate aliquots taken from each well were averaged foranalysis.

Saturation binding data were fit to a simple 1-site binding equation vianonlinear regression in Prism version 3.03 (GraphPad Software, Inc.) toobtain an apparent mean K_(D) of 46 pM for ¹²⁵I-hCSF-1 binding to cellsurface expressed human c-fms. Inhibition data were fit to a single sitecompetitive inhibition equation via nonlinear regression in Prism usingthe K_(D) value for ¹²⁵I-hCSF-1 obtained in the concurrent binding assayto generate a K_(I) value for unlabeled hCSF-1 (apparent mean K_(I)=17.8pM) as well as for each anti-c-fms mAb. Mean K_(I) values from 2experiments were reported (see, TABLE 13).

Example 11 Determination of Rate and Affinity Constants for Monomericc-fms Binding to Anti-c-fms mAbs

The affinity of human c-fms (1-512).pHIS (Amgen) for the anti-c-fms mAbswas measured by Biacore. Experiments were conducted at 25° C. using aBiacore 3000 instrument (Biacore AB) equipped with a CM4 sensor chip.Sensor chips, amine coupling reagents (EDC(1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride), NHS(N-hydroxysuccinimide), and ethanolamine-HCl, pH 8.5), 10 mM sodiumacetate, pH 5.5, HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% v/v Surfactant P20), and 10 mM glycine-HCl, pH 1.5 were purchasedfrom Biacore AB. Bovine serum albumin (BSA, Bovuminar Standard Powder)was purchased from Serological Corporation. AffiniPure Goat Anti-HumanIgG, Fcγ Fragment Specific was purchased from Jackson ImmunoResearchLaboratories.

An anti-human IgG, Fcγ specific capture antibody was covalentlyimmobilized to a CM4 chip using standard amine-coupling chemistry withHBS-EP as the running buffer. Briefly, each flow cell was activated for7 minutes with a 1:1 (v/v) mixture of 0.1 M NHS and 0.4 M EDC at a flowrate of 5 μl/min. Goat anti-human IgG at 28 μg/ml in 10 mM sodiumacetate, pH 5.5 was immobilized at a density of ˜2700 RUs. Residualreactive surfaces were deactivated with a 7 minute injection of 1 Methanolamine at 5 μl/min. Three 50 μl injections of 10 mM glycine HCl,pH 1.5 at 100 μl/min were used to remove any remaining noncovalentlybound capture antibody and to condition each surface. The running bufferwas switched to HBS-EP with 0.1 mg/ml BSA for all remaining steps.

Anti-c-fms 1.2 or 1.2 SM at 0.25 μg/ml was injected over goat anti-humanIgG, Fcγ in one flow cell for 2 minutes at 10 μl/min to obtain a surfacedensity of ˜47 RUs. Another flow cell with goat anti-human IgG, Fcγalone was used as a reference surface. Each assay started with tencycles of buffer as the analyte to stabilize the signal. Human monomericc-fms (1-512).pHIS samples were prepared at concentrations of 30, 10,3.33, 1.11, 0.37, and 0.12 nM in triplicate and injected along with 6buffer blanks in random order at 100 μl/min over both the capturedanti-c-fms and reference surfaces. Each complex was allowed to associatefor 2 minutes, and dissociate for 5 minutes. The surfaces wereregenerated after each c-fms or buffer injection with a 30-second pulseof 10 mM glycine HCl, pH 1.5 at 100 μl/min, followed by a 30-secondinjection of buffer.

Other anti-c-fms antibodies were tested in a similar manner, but changeswere made to the protocol to account for differences in bindingcharacteristics. Anti-c-fms 1.109 and 1.109 SM were each injected overgoat anti-human IgG at 0.5 μg/ml for 1.5 minutes at 10 μL/min to obtainsurface densities of 59 and 91 RUs, respectively. Human c-fms(1-512).pHIS samples were prepared at concentrations of 10, 3.33, 1.11,0.37, 0.12, and 0.041 nM for anti-c-fms 1.109 binding, and the same setwith the exception of the 0.041 nM sample was prepared for anti-c-fms1.109 SM binding. Human monomeric c-fms (1-512).pHIS was allowed todissociate from anti-c-fms 1.109 for 20 mins, and 1.109 SM for 15 mins.Anti c-fms 2.360 and 2.360 SM were each injected over goat anti-humanIgG, Fcγ at 1 μg/ml for 1.5 or 2 minutes, respectively, at 10 μl/min toobtain surface densities of ˜100 RUs. Human c-fms (1-512)/pHIS sampleswere prepared at concentrations of 30, 10, 3.33, 1.11, and 0.37 nM foranti-c-fms 2.360 binding, and the same set with the addition of a 0.12nM sample was prepared for anti-c-fms 2.360 SM binding. Human monomericc-fms (1-512).pHIS was allowed to dissociate from anti-c-fms 2.360 for 8mins, and 2.360 SM for 5 mins.

Data was double referenced by subtracting the reference surfaceresponses to remove bulk refractive index changes, and then subtractingthe averaged buffer blank response to remove systematic artifacts fromthe experimental flow cells. The data was processed and globally fit toa 1:1 interaction model with local Rmax in BIAevaluation (version 4.1,Biacore AB) to obtain kinetic rate constants k_(a) and k_(d). Thoughdata from triplicate samples at each concentration of c-fms wascollected, only data from duplicate samples could be analyzed due to theparameter number restrictions inherent to BIAevaluation software. TheK_(D) was calculated from the quotient k_(d)/k_(a). The results areshown in TABLE 19. Data from the various examples provided above aresummarized in Table 20.

TABLE 19 Binding Affinity Of Anti-c-fms mAbs To Soluble Monomeric C-fmsProtein As Measured By Biacore 3000 Antibody k_(a) (1/Ms) k_(d) (1/s)K_(D) (pM) 1.2 3.84 × 10⁶ 1.98 × 10⁻³ 516 1.2 SM* 3.62 × 10⁶ 1.99 × 10⁻³548 1.109 2.54 × 10⁶ 1.29 × 10⁻⁴ 51 1.109 SM* 2.66 × 10⁶ 2.71 × 10⁻⁴ 1022.360 1.25 × 10⁶ 6.67 × 10⁻⁴ 535 2.360 SM* 7.94 × 10⁵ 9.55 × 10⁻⁴ 1200*SM = Somatic mutations removed.

TABLE 20 Summary of mAbs 1.2, 1.109, and 2.360 Inhibition (K_(I))Binding IP from Inhibition Cynomolgus of ¹²⁵I-CSF-1 Affinity 293T Cellson Ligand AML-5 Bone Marrow Binding to (K_(D)) to Expressing InductionMonoclonal proliferation Proliferation AML-5 Cells c-fms by Wt c-fms &of pTyr of Antibody IC₅₀ (pM)* IC₅₀ (pM) (pM) Biacore (pM) SNPs c-fms1.2 27 78 8.5 516 +++ +++ 1.2 SM** 12 81 11.5 548 +++ +++ 1.109 27 1613.5 51 +++ +++ 1.109 SM** 23 9.7 102 +++ +++ 2.360 60 67 ~160 535 +++++ 2.360 SM** ~900 1200 ++ ++ *Primary cell assay on human target; **SM= Somatic mutations removed.

Example 12 Epitope Mapping of Anti c-fms Antibodies IgG₂ Clones 1.109,1.2 and 2.36

Preparation of c-fms-Avidin Fusion Constructs

The c-fms avidin fusion expression constructs are shown in FIG. 10. Toexpress each fusion protein, the coding sequence for human c-fmsextracellular domain was PCR amplified and cloned into pCEP4-Avidin(N),such that the c-fms sequence was joined to the C-terminus of the chickenavidin sequence using the restriction enzyme XhoI. The signal sequenceof c-fms was not included, as the signal sequence for chicken avidin wasleft intact in the pCEP4-avidin(N) vector.

As noted above, the extracellular domain contains five different Ig-likeregions. The different domains in human c-fms are discussed, forexample, in Hampe et al., 1989, Oncogene Res. 4:9-17. For a discussionof the corresponding domains in mouse c-fms, see, for example, Wang etal., 1993, Molecular and Cell Biology 13:5348-5359. The followingdifferent avidin constructs were prepared to correspond with theindicated Ig-like domain (see, FIG. 8 for the amino acid sequence of theextracellular domain; SEQ ID No 1).

Signal: amino acids 1-19

Ig-like 1 domain: amino acids 20-126

Ig-like 1-2 domain: amino acids 20-223

Ig-like 1-3 domain: amino acids 20-320

Ig-like 1-4 domain: amino acids 20-418

Ig-like 1-5 domain: amino acids 20-512

Ig-like 2 domain only: amino acids 85-223.

Thus, to create specific regions of human c-fms (truncations) forepitope mapping, PCR amplification was performed to target the followingamino acids: Ig-like loop1 (IPVI-ALLP), Ig-like loops 1 and 2(IPVI-AQIV), Ig-like loops 1-3 (IPVI-EGLN), Ig-like loops 1-4(IPVI-GTLL), and Ig-like loops 1-5 (IPVI-PPDE), as well as Ig-like loop2 alone (TEPG-AQIV). The sequences identified in the parenthesesindicate the starting and ending sequence respectively for each of thedomains (see FIG. 8). The particular regions indicated were selected tokeep the cysteine residues involved in disulfide bond formation, asthese bonds are important in maintaining the native three-dimensionalstructure of the domains. Furthermore, the construct for the Ig-likeloop 2 alone includes some sequence from the Ig-like loop 1 for the samereason. Consequently, the starting and ending amino acids of the domainsthat are listed differ somewhat from the domain regions specified in thearticles by Hampe and Wang listed above.

Expression of Avidin Fusion Proteins

Expression of avidin fusion proteins was achieved by transienttransfection of human 293T adherent cells in T75 tissue culture flasks.Cells were grown and maintained in DMEM (high glucose) with 10% dialyzedFBS and 1× Pen-strep-glutamine (growth medium), at 37° C. and 5% CO₂.Approximately 3×10⁶ 293T cells were inoculated into T75 flaskscontaining 15 ml of growth medium and grown overnight for approximately20 hours. Cells were then transfected with pCEP4-Avidin(N)-c-fmsconstructs. In each flask, 15 μg of DNA was mixed with 75 μl ofLipofectamine 2000 (Invitrogen) in the presence of Opti-MEM medium(Invitrogen) and the complex was incubated for 20 minutes. Thetransfection complex was inoculated into the corresponding flask andincubated at 37° C. for 4-5 hours in Opti-MEM media. At the end of theincubation time, the Opti-MEM medium was replaced with fresh growthmedium. Approximately 48 hours post-transfection, the conditioned mediawas harvested and centrifuged at 2000×g for 10 minutes at 4° C. toremove cells and debris, and transferred to 50 ml tubes. A control flaskwas also made following the same protocol, but no DNA was used (mocktransfection), yielding negative control conditioned media for bindingexperiments.

Detection of Fusion Proteins

The concentration of each c-fms avidin fusion protein was determinedusing a quantitative FACS based assay. The c-fms avidin fusion proteinswere captured on 6.7 μm biotin polystyrene beads (Spherotech, Inc.,Libertyville Ill.). 1× conditioned media (20 and 200 μl) were added to 5μl (˜3.5×10⁵) beads, and incubated for 1 hr at room temperature withrotation. Conditioned media was removed by centrifugation and sampleswere washed with PBS containing 0.5% BSA (BPBS). The avidin beads werestained with 200 μl of a 0.5 μmg/ml solution of a goat FITC-labeledanti-avidin antibody (Vector Labs, Burlingame, Calif.) in BPBS for 45minutes at room temperature covered by foil. Following incubation, thebeads were recollected by centrifugation, washed with BPBS, andresuspended for analysis in 0.5 ml BPBS. The FITC fluorescence wasdetected using a FACScan (Becton Dickinson Bioscience, Franklin Lakes,N.J.). The signal was converted to protein mass using a standard curvederived with rAvidin. For epitope mapping, biotin beads were loaded with˜100 ng of c-fms avidin fusion protein per 3.5×10⁵ beads and brought upto volume with growth medium.

Antibody Binding FACS Assay

Biotin-coated polystyrene beads (Spherotech, Inc.) loaded withnormalized amounts of C-FMS subdomain fusion proteins were mixed with 1mg of FITC conjugated anti c-fms monoclonal antibody (1.109, 1.2 and2.36) in 0.2 ml of BPBS. After incubation for 1 hr at room temperature,3 ml washing buffer (BPBS) was added and the antibody-beads complexeswere collected by centrifugation for 5 min at 750×g. The pellet waswashed in 3 ml of BPBS. The antibody bound to avidin-bead complexes wasdetected by FACS (Becton Dickinson) analysis. The mean (X) fluorescentintensity was recorded for each sample.

Antibody Competition Assay

To prepare for labeling with fluorescein, the monoclonal antibodies weredialyzed or resuspended at a concentration of 1 mg/ml in PBS (pH 7.4).Label ([6-fluorescein-5-(and -6)-carboxamido]hexanoic acid, succinimidylester 5(6)-SFX) mixed isomers from Molecular Probes (F-2181) was addedto the protein at a molar ratio 9.5:1 (label:protein) from a label stockof 5 mg/ml in DMSO. The mixture was incubated at room temperature for 1hour in the dark. The labeled antibody was separated from the free labelby dialysis in PBS. For each competition experiment, a binding reactionwas assembled that contained a 20-fold excess (20 μg/ml) of unlabeledcompetitor antibody, 3.5×10⁵ biotin beads coated with the avidin fusionprotein in BPBS. The FITC-labeled antibody (1 μg/ml) was added after a30 min pre-incubation of unlabeled competitor antibody. The processfollowed the one color method from this point forward.

All the fusion proteins (FIG. 11) expressed in 293T cells can bedetected with FITC-labeled anti-avidin antibody by FACScan. To determinewhich c-fms Ig-like domain is the antibody-binding site, all six fusionproteins were used in a binding assay. The antibody clones 1.109, 1.2and 2.36 bind to the human c-fms subdomain Ig-like1-2, Ig-like1-3,Ig-like1-4 and Ig-like1-5 fusion proteins. They do not bind to thesingle domain c-fms Ig-like1 and Ig-like2 fusion proteins. Forcomparison, human c-fms ECD is used as a positive control (FIGS. 11 and12). These results indicate that the epitopes of these three antibodiesare mainly located at the N-terminus Ig-like loop1 and Ig-like loop2 ofhuman c-fms, and require the presence of both the Ig-like loop 1 and theIg-like loop 2 regions. The results also indicate that the antibody maynot directly block the high-affinity binding site of the ligand which ismainly located at Ig-like loop3. It may indirectly affect the ligandbinding due to Ig-like loops 1 and 2, both of which are critical regionsfor ligand binding (Wang, et al., 1993, Molecular Cell Biology13:5348-5359).

Among the three antibodies, clone 1.109 has the highest binding signalcompared to the other two antibodies under 1 μg/ml. The competition datademonstrated that three of the antibodies can block each other with20-fold excess unlabeled antibody (see, FIGS. 13, 14 and 15). Thecompetition data also indicate that the epitope of these threeantibodies are similar or adjacent within Ig-like loops 1 and 2.

Example 13 Epitope Mapping of Anti-c-fms Antibody 1.2 SM VersusCommercial Antibodies

This experiment was conducted to determine if certain of the humanantibodies disclosed herein bound the same or a different epitope than anumber of commercially-available antibodies.

Materials and Methods: Commercial c-fms Antibodies Tested

Rat and mouse antibodies tested are shown in Table 21 and Table 22.

TABLE 21 Rat antibodies Cat. No. and Shorthand Binding region per SourceClone No. Designation manufacturer Biosource Cat. No. AHT1512, 2-4A5-2Between amino acids clone 2-4A5-2 349-512 US Cat. No. C2447-53, 5J15Between amino acids Biological clone 5J15 349-512 US Cat. No. C2447-50,0N178 Not described Biological clone O.N. 178

TABLE 22 Mouse Antibodies Cat. No. and Shorthand Binding region perSource Clone No. Designation manufacturer R&D Cat. No. MAB329, MAB329Extracellular domain Systems clone 61708 used as immunogen R&D Cat. No.MAB3291, MAB3291 Extracellular domain Systems clone 61701 used asimmunogen R&D Cat. No. MAB3292, MAB3292 Extracellular domain Systemsclone 61715 used as immunogen

c-fms-Avidin Fusion Constructs and Expression of Avidin Fusion Proteins

Human c-fms avidin fusion expression constructs were prepared asdescribed in Example 12. Expression of avidin fusion proteins wasachieved by transient transfection of human 293T adherent cells in 10 cmtissue culture plates. Cells were grown and maintained in DMEM (highglucose) containing 5% qualified FBS and supplemented with 1×Pen-strep-glutamine (Invitrogen), 1× non-essential amino acids(Invitrogen) and 1× sodium pyruvate (Invitrogen) (growth medium), at 37°C. and 5% CO₂. Approximately 2.5×10⁶ 293T cells were inoculated into 10cm plates containing 10 ml of growth medium and grown overnight forapproximately 20 hours. Cells were then transfected withpCEP4-Avidin(N)-c-fms constructs. For each transfection, 7.5 μg of DNAwas mixed with 45 μl of FuGene6 (Roche) in the presence ofsupplement-free DMEM medium (Invitrogen) and the complex was incubatedfor 20 minutes. The transfection complex was added to the correspondingplate and incubated at 37° C. overnight. The following morning, thecells were washed twice with 1× Dulbecco's Phosphate Buffered Saline(PBS) (Invitrogen) and fed with 5 ml of serum free DMEM containing theaforementioned supplements plus Insulin, Transferrin, Selenium-X (ITS-X)(Invitrogen). Approximately 48 hours post-transfection, the conditionedmedia was harvested and centrifuged at 2000×g for 10 minutes at 4° C. toremove cells and debris, and transferred to 15 ml tubes. A control platewas also made following the same protocol, but no DNA was used (mocktransfection), yielding negative control conditioned media for bindingexperiments.

Detection of Fusion Proteins

The concentration of each c-fms avidin fusion protein was determinedusing a quantitative FACS based assay. Avidin fusion proteins werecaptured on 6.7 μm biotin polystyrene beads (Spherotech, Inc.,Libertyville Ill.). 1× conditioned media (2, 20 and 200 μl) were addedto 5 μl (˜3.5×10⁵) beads, and incubated for 1 hr at room temperaturewith rotation. Conditioned media was removed by centrifugation andsamples were washed with PBS containing 2% FBS (FPBS). The avidin beadswere stained with 500 μl of a 1.0 μg/ml solution of a FITC-labeled goatanti-avidin antibody (Vector Labs, Burlingame, Calif.) in FPBS for 30minutes at room temperature with rotation. Following incubation, thebeads were recollected by centrifugation, washed with FPBS, andresuspended for analysis in 0.5 ml FPBS. The FITC fluorescence wasdetected using a FACScan (Becton Dickinson Bioscience, Franklin Lakes,N.J.). The signal was converted to protein mass using a standard curvederived with rAvidin. For epitope mapping, biotin beads were loaded with˜200 ng of c-fms avidin fusion protein per 3.5×10⁵ beads and brought upto volume with FPBS.

Antibody Binding FACS Assay

Biotin-coated polystyrene beads (Spherotech, Inc.) loaded withnormalized amounts of c-fms subdomain fusion proteins were mixed with 1μg of either human anti c-fms monoclonal antibody (1.2) mouse anti-c-fmsmonoclonal antibody (MAB 329, MAB 3291 and MAB3292 [R&D Systems]) or ratanti-c-fms monoclonal antibody (2-4A5-2 [Invitrogen], O.N.178 and 5J15[U.S. Biological]) in 0.2 ml of FPBS. After incubation for 1 hr at roomtemperature, the antibody-bead complexes were washed three times with1.25 ml washing buffer (FPBS) with collection by centrifugation for 1min at 18,000×g between washes. The antibodies were then stained aspecies appropriate goat secondary antibody conjugated to FITC (SouthernBiotech) at 1.0 μg/ml for 30 min. The wash steps were repeated and theantibody-bead complexes were resuspended in 0.5 ml FPBS for analysis.The antibody bound to avidin-bead complexes was detected by FACS (BectonDickinson) analysis. The mean (X) fluorescent intensity was recorded foreach sample.

Antibody Competition Assay

To prepare for labeling with fluorescein, the monoclonal antibodies weredialyzed or resuspended at a concentration of 1 mg/ml in PBS (pH 7.4).Label ([6-fluorescein-5-(and -6)-carboxamido]hexanoic acid, succinimidylester 5(6)-SFX]) mixed isomers from Molecular Probes (F-2181) was addedto the protein at a 10:1 molar ratio (label:protein) from a stock of 10mg/ml in DMSO. The mixture was incubated at room temperature for 1 hourin the dark. The labeled antibody was separated from the free label byNAP 5 column chromatography in PBS followed by 0.2 μm filtration. Foreach competition experiment, a binding reaction was assembled thatcontained a 25-fold excess (25 μg/ml) of unlabeled competitor antibody,3.5×10⁵ biotin beads coated with the avidin fusion protein in FPBS. TheFITC-labeled antibody (1 μg/ml) was added after a 15 min pre-incubation.The process followed the one color method from this point forward.

Results and Discussion

All the fusion proteins expressed in 293T cells can be detected withFITC-labeled anti-avidin antibody by FACScan. As described in Example12, several antibodies that were tested bind similar epitopes thatrequire the presence of both Ig-like loop 1 and Ig-like loop 2 regionsfound in the Ig1-2 avidin fusion construct. Consequently, binding andcompetition experiments were done with one of the human antibodiesprovided herein, the commercially available anti-human c-fms antibodiesand select members of the panel of avidin fusion constructs.

All of the commercial antibodies were able to successfully bind to thefull length c-fms ECD Ig1-5 construct as expected. Of the six commercialantibodies, one (MAB 3291) was able to bind to the Ig1-2 construct,indicating a possible competitor for the human anti-c-fms epitopes.Further binding experiments were done using the Ig1 construct. MAB3291was shown to bind the Ig1 construct, indicating that its epitope waslocated entirely within the Ig1-like domain. The slight signal seen forMAB329 in the Ig1 and Ig1-2 constructs was confirmed to be backgroundbinding of the antibody to the beads.

The competition data demonstrated that none of the commercially sourcedantibodies can block the representative human antibody even at a 25 foldexcess of competitor antibody.

The combined data from the binding and competition experimentsdemonstrate that the commercial antibodies bind to epitopes which arenot utilized by the human anti-c-fms antibodies.

Example 14 Epitope Mapping of Anti c-fms Antibodies by Arginine/GlutamicAcid Scanning of c-fms

An arginine/glutamic acid-scanning strategy was used to map antibodybinding to c-fms. The arginine and glutamic acid sidechains are chargedand bulky, and may disrupt antibody binding to c-fms. This method canthus indicate residues that when mutated negatively affect the bindingof the antigen binding protein to c-fms. This indicates that thecorresponding residues in the unmutated antigen binding protein can bein contact with the antigen binding protein or in close proximity to theantibody such that substitution with arginine or glutamic acid issufficient to affect binding.

Construction, Expression, and Characterization of Arginine/Glutamic AcidMutants

Ninety-five amino acids distributed throughout the first three Igdomains of c-fms were selected for mutation. The selection was biasedtowards charged or polar amino acids, excluding cysteine and proline inorder to reduce the likelihood of the mutation resulting in a misfoldedprotein. Non-arginine amino acids were mutated to arginine; arginine andlysine were mutated to glutamic acid.

Sense and anti-sense oligonucleotides containing the mutated residueswere synthesized in a 96-well format. Mutagenesis of the c-fmsextracellular domain-Flag-His-tagged construct (“wild type”) wasperformed using a Quikchange II kit (Stratagene, #200523). All mutantconstructs of Flag-His-tagged c-fms in the pTT5 vector, were expressedin transiently transfected 293-6E suspension cells (NRCC) in 96-wellplates. Expression levels and integrity of the recombinant protein inconditioned media were characterized by western blot against the His-tagfollowed by an anti-isotype Alexa-fluor antibody. Subsequent epitopemapping experiments were performed using protein in conditioned media.

Mutant expression was characterized by running supernatants from eachwell on an ePage SDS-PAGE electrophoresis apparatus (Invitrogen),blotting, and probing with an anti-His antibody (Novagen) followed by ananti-isotype Alexa-fluor antibody. Each mutant construct was expressed.

Determination of Conformational Epitopes

To determine whether anti-c-fms antibodies bound to a conformationalepitope on c-fms, three anti-c-fms antibodies (1.2 SM, 1.109 SM and2.360) and c-fms were individually run on western blots under reducingand non-reducing conditions. Antibodies 1.2 SM and 2.360 were shown tobind a conformational epitope as evidenced by the lack of bands inwestern blots under reducing conditions, whereas a light band wasobserved with antibody 1.109 SM, indicating that it can bind a linearepitope.

BioPlex Binding Assay

A bead-based multiplexed assay was used to measure antibody binding tothe 95 c-fms mutants, wild type, and a negative control simultaneously.One hundred sets of color-coded strepavidin-coated LumAvidin beads(Luminex) were bound with biotinylated anti-pentaHis antibody (Qiagen,#1019225) for 1 hour at room temperature (RT) then washed. Eachcolor-coded bead set was then allowed to bind to a c-fms mutant,wild-type, or negative control in 100 μl supernatant for 1 hour at RTand washed.

The color-coded bead sets, each associated to a specific protein, werepooled. The pooled beads were aliquoted to 96 wells of a 96-well filterplate (Millipore, #MSBVN1250). 100 μl of anti-c-fms antibodies (1.2 SM,1.109 SM and 2.360) in 3-fold dilutions were added to three columns fortriplicate points and incubated for 1 hour at RT and washed. 100 μl of1:200 dilution phycoerythrin (PE)-conjugated anti-human IgG Fc (JacksonImmunoresearch, #109-116-170) was added to each well and incubated for 1hour at RT and washed.

Beads were resuspended in 1% BSA in PBS, shaken for 10 minutes and readon the BioPlex instrument (Bio-Rad). The instrument identified each beadby its color-code and measured the amount of antibody bound to the beadsaccording to the fluorescent intensity of the PE dye. Antibody bindingto each mutant was compared directly to its binding to the wild type inthe same pool.

Identifying Antibody Binding to Mutant c-fms

The variability of the assay system and significance of changes inbinding were determined empirically. Bead-to-bead and well-to-wellvariability was experimentally determined by binding wild-type c-fms toall 100 sets of color-coded beads. Beads were dispensed to each well ofa 96-well plate and probed with anti-c-fms antibody 1.2 SM in 3-folddilutions down each column of the plate, across all 12 columns of theplate. EC50 were derived using curve fits from measuring the variabilityof maximum signals, minimum signals and slope. Variability measurementswere used to determine whether a magnitude shift in EC50s wassignificant.

Mean fluorescence intensity (MFI) of antibody binding was graphed usinga weighted 4 Parameter Logistical curve fit (4PL with VarPower inSplus). Experimental variability was determined using three wild typecontrols in each pool. Antibody binding to mutant antigen was comparedto each wild type control. A 99% confidence interval (CI) of the EC50fold change between mutant and each wild type control was calculated andthe comparison to the wild type control giving the larger p-value wasreported. Multiplicity adjustment using Benjamini-Hochberg FalseDiscovery Rate (FDR) control was applied. Mutations whose 99% CI of theEC50 is significantly different from wild type EC50, that is having anFDR adjusted p-value of 0.02 or less, were considered important in thespecific binding reaction between the protein antigen and antigenbinding protein. In addition, mutations that reduced binding asevidenced by a reduction in maximum MFI signal to 30% or less of wildtype were considered to significantly influence binding between theprotein antigen and antigen binding protein. Table 23 summarizes the“hits” or the position of mutations that significantly reduced theability of the 1.2 SM, 1.109, and 2.36 antibodies to bind theextracellular domain of human c-fms. The notation used in Table 23 is:(wild-type residue:position in polypeptide:mutant residue), where thenumbering is as shown in SEQ ID NO:1.

TABLE 23 Summary of mutations that affect antibody binding. AntibodyHits 1.2 SM E29R, Q121R, T152R, K185E 1.109 E29R, Q121R, S172R, G274R,Y276R 2.36 R106E, H151R, T152R, Y154R, S155R, W159R, Q171R, S172R,Q173R, G183R, R184E, K185E, E218R, A220R, S228R, H239R, N240R, K259E,G274R, N275R, Y276R, S277R, N282R ALL EC50 shift K102E, R144E, R146E,D174R, A226R low/no W50R, A74R, Y100R, D122R, T130R, binding G161R,Y175R, A179R

Because binding of at least one antibody is maintained in the presenceof the particular mutations shown in Table 23, the mutant proteins areunlikely to be grossly misfolded or aggregated due to the introducedmutation. This is also true for those mutations which caused an EC50shift for all of the antibodies as the antibodies are still able to bindantigen. Although each of the tested antibodies bind to a similar regionas shown by the binning analysis, each antibody can be distinguished bythe mutations which inhibit antibody binding to mutant antigen. Thatsome of the mutations affect multiple antibodies is consistent with thefact that the antibodies belong to similar bins.

Example 15 Inhibition of the Growth of MDAMB231 Breast AdenocarcinomaXenograft

Because the antigen binding proteins provided herein bind human c-fmsbut not mouse c-fms, a series of in vivo experiments were conducted withan antibody that binds murine c-fms to demonstrate the utility of ananti-cfms antibody to treat cancer.

Athymic nude mice were subcutaneously implanted with 10 million MDAMB231human breast adenocarcinoma cells in the presence of Matrigel (1:1).Starting within one day of tumor cell implantation, mice were injectedintraperitoneally with either 400 μg of anti-murine c-fms antibody or400 μg of control rat anti-mouse IgG in 100 μl PBS 3 times per week forthe duration of the study. Tumor measurements and treatment days areshown in the FIG. 16. After 51 days mice were euthanized and tumorscollected and formalin fixed. H&E stained sections and F4/80 (macrophagemarker) targeted immunohistochemistry sections were evaluated. Allscoring was done blinded to treatment and group. Sections from micetreated with anti-murine c-fms antibody showed significantly lessstaining than mice treated with the control, thus indicating asignificant reduction in the number of tumor associated macrophages. Tomore objectively evaluate the extent of necrosis, digital images of AFOGstained sections were captured using Metavue software, the entirecross-sectional area and necrotic cross-sectional area of tumors weremeasured. The percent necrosis of each tumor was then calculated fromthese measurements and shown in FIG. 16. These results demonstrate thatan anti-c-fms antibody can decrease tumor associated macrophages,increase necrosis and inhibit the growth of MDAMB231 breastadenocarcinoma xenografts.

Example 16 Inhibition of the Growth of Established NCIH1975 LungAdenocarcinoma Xenograft

Athymic nude mice were subcutaneously implanted with 10 million NCIH1975human lung adenocarcinoma cells in the presence of Matrigel (1:1). Aftertumors were allowed to grow to 250-300 mm³, mice were injectedintraperitoneally with either 400 μg of anti-murine c-fms antibody or400 μg of control Rat anti-mouse IgG in 100 μl PBS 3 times per week forthe duration of the study. A third group of mice was treated with 30mg/kg Taxotere (positive control) once per week. Tumor measurements andtreatment days are shown in FIG. 17 which shows that an anti-cfmsantibody can inhibit the growth of an established NCIH1975 lungadenocarcinoma xenograft.

The above tumor models demonstrate the utility of an anti-cfms antibodythat inhibits the activity of the CSF-1/cfms axis, such as thosedisclosed herein, for use in the treatment of cancer. The ability ofsuch antibodies to decrease infiltrating macrophages in diseased tissuemeans that the antibodies can also be used in metabolic and inflammatorydiseases.

Example 17 Modulation of the CSF-1/CSF-1R Interaction to ControlAngiogenesis

To test whether CSF-1 mediated recruitment, differentiation andstimulation of macrophages may be involved in promoting angiogenesis intumors or other normal tissues, two different neutralizing ratanti-murine CSF-1R monoclonal antibodies (M279 was generated internally;the other, FS98, was obtained from Ebiosciences), were evaluated fortheir effect on mouse corneal angiogenesis in vivo. On day five after asingle systemic dose of control mAb, M279 or AFS98, the followingparameters were measured and analyzed: 1) the vascular densityassociated with the mouse corneal angiogenesis response, 2) circulatinglevels of mouse CSF-1, and 3) levels of macrophage infiltration in thecornea and other tissues.

Mouse Corneal Pocket Assay

A 4 mm PVA sponge (M-PACT Worldwide, Eudora, Kans.) was precisely cutinto two equal pieces and immersed in 8 μL1 of PBS containing 2.4 μg ofrecombinant human FGF-2 or 48 μg of recombinant human VEGF (R&D Systems,Minneapolis, Minn.). The sponge was further asceptically processed into48 similarly sized mini-sponge fragments (pellets) suitable for cornealpocket implantation. Each sponge fragment contained approximately 50 ngof recombinant human FGF-2 or 1 μg of recombinant human VEGF. Female C57BL/6 mice (7-10 weeks of age) were anesthetized using systemicanesthesia and eyes prepared for corneal incision by placing a singledrop of Proparacaine topical anesthetic in each eye. A fine slit wascreated in the middle of the cornea and an opening (“pocket”) created inthe corneal stroma that followed the curvature of the eye approximately1 mm from the limbus. Pellets containing PBS, VEGF or FGF-2 were placedinto the corneal pockets and animals treated with either rat IgG (Sigma,St. Louis, Mo.) IP at a dose of 250 μg in 200 μl pyrogen-free PBS, orwith 250 μg purified rat anti-mouse CSF-1R. On day 5 post pelletimplantation, mice were anesthetized with systemic anesthesia and eacheye (cornea) imaged under a stereomicroscope fitted with an Insight Spotdigital camera fitted with a near vertical illuminator at an incipientangle of 45 degrees from the polar axis in the meridian containing thepellet. These acquired digital images were processed by subtractivecolor filters (Adobe Photoshop) and analyzed using Bioquant imageanalysis software (Nashville, Tenn.) to determine the fraction of pixelswithin the total density of the corneal perimeter that exceeded athreshold matching visible capillaries. Total vascular density of thecornea was determined by using the fraction of pixels, the result ofwhich was expressed as a ratio of blood vessel area pixel number towhole eye area pixel number.

Mouse CSF-1 ELISA

Serum levels of mouse CSF-1 were determined as a biomarker of antiCSF-1R antibody activity using the DUOSET antibody ELISA system (R&Dsystems) according to the manufacturer's instructions.

Immunolocalization of Macrophage and Blood Vessels in Liver and CornealTissues of Mice

To determine the effects of anti-mouse CSF-1R antibodies on macrophageand blood vessel levels in tissue, rat anti-mouse F4/80 (amacrophage-restricted cell surface glycoprotein), conjugated with Alexa488, clone BM8 (1:1000) was used to detect tissue macrophages. CD31, ratanti-mouse PECAM-1 IgG2a, conjugated with PE, clone 390 (use 1 μg/ml)was used to detect endothelial cells. After tissue was harvested, it wasfrozen in OCT for further processing. 5 micron sections of either liveror cornea were fixed with cold acetone for 15 min at room temperatureand then washed twice with PBS. After washing, sections were incubatedin blocking solution (BS) for 30 min at room temperature. Both F4/80-488and CD31-PE were added at the above concentrations in BS and sectionsincubated 30 minutes at room temperature followed by twice washing withPBS. Slides were mounted in mounting media and fluorescent imagesacquired with Leica-Hamamutsu-Openlab system.

Results and Conclusion:

Rat anti-muCSF-1R neutralizing antibodies, M279 and AFS98, significantlyinhibited FGF-2, but not VEGF-induced mouse corneal angiogenesis byapproximately 80% (P<0.01). A single dose of 250 μg M279 or AFS98,significantly increased muCSF-1 serum levels compared to levels observedin rat IgG-treated mice (45-83 fold increase) Immunofluorescentstaining/localization (IMF) results in mouse corneal sections showedthat FGF-2 and VEGF pellet implantation increases macrophageinfiltration in the cornea compared to surgery/PBS pellet implantationalone. M279 treatment robustly diminished stimulus-induced (both FGF-2and VEGF) corneal macrophage infiltration compared to control rat IgGtreatment by approximately 85 to 96 percent. The IMF results in mouseliver sections also showed that the single treatment with M279 or AFS98significantly decreased the number of F4/80 positive macrophages byapproximately 60% in the mouse liver while not appreciably alteringvascular density as assessed by CD31 IMF (P<0.01). Macrophages followthe blood vessel network but do not generally co-localize withmicrovessels in the vascularized mouse cornea.

When evaluating both angiogenic stimuli (FGF-2 and VEGF), blocking theCSF-1/CSF-1R interaction decreased macrophage infiltration to the tissuewhile it only inhibited FGF-2 angiogenesis based on corneal vesseldensity imaging. Based on these results, it appears that theinflammatory environment dictates when CSF-1 responsive tissuemacrophages can facilitate/promote angiogenesis, while at the same timeillustrating that tissue macrophages at multiple inflammatory sitesrequire ongoing CSF-1/CSF-1R interaction to maintain their presence atthe inflammatory lesion. The results indicate that in cases whereinflammatory angiogenesis is driven primarily by FGF-2 that inhibitingthe CSF-1/CSF-1R interaction can be beneficial in decreasing new bloodvessel formation, especially in tumors where VEGF levels are not highbut tumor vascular density is.

Example 18 Toxicology Studies in Cynomolgus Monkeys

Cynomolgus monkeys were administered the 1.2 SM antibody andpharmocodynamic markers were measured. The cohort used to study theeffects of a c-fms antigen binding protein is shown in TABLE 24.Antibody 1.2 SM was administered weekly by intravenous injection toCynomolgus monkeys for 4 weeks followed by an 11-week recovery period,with terminal necropsy on day 29 and recovery necropsy at 3 months.Pharmocodynamic markers including serum CSF-1 levels, Tartrate-resistantacid phosphatase 5b (Trap5b) concentrations and the quantity of colonmacrophages were measured. As described in greater detail below, themeasurement of each of these markers demonstrated the ability of theantibody to bind c-fms and inhibit the c-fms/CSF-1 axis. The level ofthe markers also correlated with the level of antibody in the blood.

TABLE 24 Cohort for toxicology study No. M/F No. M/F Dose No. Males/Terminal Recovery Group (mg/kg) Females Necropsy Necropsy 1 0 5/5 3/32/2 2 20 5/5 3/3 2/2 3 100 5/5 3/3 2/2 4 300 5/5 3/3 2/2

Response of Serum CSF-1 Levels to Treatment with Antibody 1.2 SM

Serum CSF-1 levels provide a biomarker for the presence and activity ofanti-c-fms antibody. This is evidenced by the selective degradation bymacrophage of ¹²⁵I-labeled CSF-1 in mice (Tushinski R J et al. Cell(1982) 28:71-81); observations that CSF-1 is elevated in serum of thec-fms knock out mice (Dai X M et al. Blood (2002) 99:111-120); anddemonstrations that serum CSF-1 levels are elevated in mice treated withan anti-mouse c-fms antibody.

Relative concentrations of Cynomolgus CSF-1 were determined for serumspecimens collected at −7, 8, 29, 57, 85, and 99 days. Samples wereanalyzed using an enzyme-linked immunosorbent assay (ELISA) followingthe protocol provided by the assay manufacturer (R&D Systems Human CSF-1DuoSet ELISA kit; Minneapolis, Minn.). Cynomolgus CSF-1 concentrationswere determined by comparison to a human CSF-1 standard curve.

To measure the concentration of serum antibody 1.2 SM, a mouse anti-1.2SM antibody was passively adsorbed to Maxisorp microplate wells (Nunc).The microplate wells were blocked with SuperBlock®T20 (Pierce, Rockford,Ill.) after removing excess mouse anti-1.2 SM antibody. Standards andquality controls (QCs) were prepared by spiking antibody 1.2 SM into100% Cynomolgus monkey serum pool. The microplate wells were washedfollowing blocking. The standards, matrix blank (NSB), QCs, and studysamples were thawed at ambient room temperature then loaded into themicroplate wells after pretreating 1/50 with SuperBlock®T20. Theantibody 1.2 SM in the samples was captured by the immobilized mouseanti-1.2 SM antibody. Unbound antibody 1.2 SM was removed by washing themicroplate wells. Following washing, a second horseradish peroxidase(HRP) conjugated mouse anti-1.2 SM antibody was added to the microplatewells to bind the captured antibody 1.2 SM. Unbound HRP conjugatedantibody was removed by washing. A 2-component3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added to thewells. The TMB substrate solution reacted with peroxide, and in thepresence of HRP created a colorimetric signal that was proportional tothe amount of antibody 1.2 SM bound by the capture reagent in theinitial step. The color development was stopped and the intensity of thecolor (optical density, OD) was measured at 450 nm to 650 nm. Data wasreduced using Watson version 7.0.0.01 reduction package using a Logistic(auto-estimate) (4 parameter logistic; 4-PL) regression model with aweighting factor of 1/Y.

Treatment of monkeys with antibody 1.2 SM resulted in a significantincrease in serum CSF-1 levels and this correlated with the antibodyserum concentration. Thus, CSF-1 serum levels were found to increaseafter administration of antibody and its accumulation in the serum andthen to decrease after administration was completed and the serumantibody levels decreased. These observations are consistent with theantibody acting on the c-fms/CSF-1 axis.

Response of Trap5b Levels to Treatment with Antibody 1.2 SM

Trap5b levels provide a marker for anti-c-fms antibodies. Trap5b isspecifically expressed by osteoclasts and is an indicator of osteoclastnumber. Osteoclasts, which are derived from the myeloid lineage ofhematopoietic cells express c-fms and utilize CSF-1. Consistent withthis is the observation that loss of CSF-1 results in decreasedosteoclasts and levels of Trap5b in CSF-1 knock out (op/op) mice (Dai XM et al., Blood (2002) 99:111-120).

The BoneTRAP® assay (Immunodiagnostic Systems Limited, Fountain Hills,Ariz.) was used to quantitate TRAP5b in subjects. Antibody 1.2 SM serumconcentrations were determined as described above. Levels of TRAP5b andantibody 1.2 SM in serum were determined for serum specimens collectedat −7, 8, 29, 57, 85, and 99 days.

At day 29 of the dosing phase, all subjects treated with 1.2 SM antibodyhad decreased Trap5b. Subsequent to treatment with antibody 1.2 SM,serum Trap5b concentrations increased as serum antibody concentrationsdecreased. Treatment with antibody 1.2 SM correlated with a decreasedserum Trap5b concentration.

Response of Macrophage to Treatment with Antibody 1.2 SM

As an additional indicator of the activity of antibody 1.2 SM inCynomolgus monkeys, the number of macrophages present in colon tissuewas quantitated by laser scanning cytometry (LSC) of CD-68-stainedtissue. Colon samples were collected from 3 animals/sex/group at the day29 necropsy and 2 animals/sex/group at the day 100 necropsy. A sample ofeach tissue was collected in OCT (Optimal Cutting Temperature) media(Sakura Finetek, Torrance, Calif.) and frozen in a dry ice/butane bath.Macrophages were stained using conventional immunohistochemistry withanti-CD68 or an isotype antibody. Diaminobenzidine (DAB) positive eventswere enumerated using laser scanning cytometry (LSC). A 2 scan methodwas used in which laser light absorption was quantified by thephotodiode detector above the stage. The first low resolution passidentified the position of the section on the slide and the subsequenthigh resolution pass acquired field images. Quantitative analysis of DABstaining was performed using the LSC-associated software.

TABLE 25 summarizes the changes in the number of colon macrophageimmediately after treatment (Day 29) and subsequent to a recovery periodwhere antibody 1.2 SM was no longer administered (Day 99). As can beseen from this table, administration of antibody 1.2 SM reduced thenumber of colony macrophage, whereas the number of macrophage increasedafter treatment was discontinued, thus demonstrating the activity of theantibody.

TABLE 25 Effect of Antibody 1.2 SM on Macrophage Population GroupComparison ANOVA Dose Group % decrease p-values Day Effect p-value(mg/kg) mean (vs. Control) (vs. Control) 29 gender 0.4461 0 11.05 dose0.0005 20 1.75 84.16% 0.0013 100 2.36 78.64% 0.0037 300 1.94 82.44%0.0003 99 gender 0.8172 0 10.62 dose 0.1043 20 12.12 −14.12% 0.9953 1009.23 13.09% 0.8493 300 2.67 74.86% 0.0975

Example 19 Treatment of a Tumor in a Patient Using a c-fms BindingProtein

A human patient is diagnosed with a malignant tumor. The patient istreated with an effective amount of a c-fms binding protein describedherein. Subsequent to administration of the c-fms binding protein, thesize and/or metabolic activity of the tumor is measured (e.g., by MRI orPET scans). Significant reductions in the size and/or metabolic activityor other indicators of tumor growth, viability, metastasis are found inresponse to administration of the c-fms binding protein.

Example 20 Reduction of TAMs in a Patient with a Malignant Tumor Using ac-fms Binding Protein

A human patient is diagnosed with a malignant tumor. The patient istreated with an effective amount of a c-fms binding protein describedherein. Subsequent to administration of the c-fms binding protein, thenumber of TAMs is measured. Significant reductions in the number of TAMsare found in response to administration of the c-fms binding protein.

Example 21 Treatment of Cachexia Using a c-fms Binding Protein

A human patient is diagnosed with cancer. The patient is treated with aneffective amount of a c-fms binding protein described herein. Subsequentto administration of the c-fms binding protein, the level of cachexia isassessed. A significant reduction in the level of cachexia is found inresponse to administration of the c-fms binding protein.

Example 22 Reduction of Vascularization Using a c-fms Binding Protein

A human patient is diagnosed with a malignant tumor. The patient istreated with an effective amount of a c-fms binding protein describedherein. Subsequent to administration of the c-fms binding protein, abiopsy of the tumor is taken and the level of vascularization isassessed. A significant reduction in the level and/or function of tumorvascularization is found in response to administration of the c-fmsbinding protein.

Example 23 Treatment of Inflammatory Arthritis Using a c-fms BindingProtein

A human patient is diagnosed with inflammatory arthritis. The patient istreated with an effective amount of a c-fms binding protein describedherein. Subsequent to administration of the c-fms binding protein, thelevel of inflammation and/or bone density is assessed. A significantreduction in the level of inflammation and/or bone osteolysis is foundin response to administration of the c-fms binding protein.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the described. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

What is claimed is:
 1. A method for treating cancer in a cancer patientcomprising administering an effective amount of an antibody or antibodyfragment to the patient, wherein (a) the antibody or antibody fragmentbinds human c-fms and comprises: (i) a heavy chain variable domaincomprising a CDRH1, a CDRH2 and a CDRH3, wherein the CDRH1 comprises theamino acid sequence of SEQ ID NO:147, the CDRH2 comprises the amino acidsequence of SEQ ID NO:163, and the CDRH3 comprises the amino acidsequence of SEQ ID NO:186; and (ii) a light chain variable domaincomprising a CDRL1, a CDRL2 and a CDRL3, wherein the CDRL1 comprises theamino acid sequence of SEQ ID NO:193; the CDRL2 comprises the amino acidsequence of SEQ ID NO:214 and the CDRL3 comprises the amino acidsequence of SEQ ID NO:228; and (b) the cancer is selected from the groupconsisting of breast cancer, prostate cancer, endometrial cancer,bladder cancer, kidney cancer, esophageal cancer, squamous cell cancer,uveal melanoma, follicular lymphoma, renal cancer, cervical cancer,gastric cancer, astrocytic cancer, lung cancer, ovarian cancer, andcolorectal cancer.
 2. The method of claim 1, wherein the antibody orfragment has one or more of the following characteristics: (a) is amonoclonal antibody; (b) is a chimeric, a humanized or a fully humanantibody; (c) is an antibody of the IgG1-, IgG2-, IgG3- or IgG4-type;(d) is a Fab fragment, a Fab′ fragment, an F(ab′)2 fragment, or an Fvfragment.
 3. The method of claim 1, wherein the cancer is selected fromthe group consisting of breast cancer, prostate cancer, lung cancer,ovarian cancer, and colorectal cancer.
 4. A method for treating cancerin a cancer patient comprising administering an effective amount of anantibody or antibody fragment to the patient, wherein (a) the antibodyor antibody fragment binds human c-fms and comprises: (i) a heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO:77; and(ii) a light chain variable domain comprising the amino acid sequence ofSEQ ID NO:110; and (b) the cancer is selected from the group consistingof breast cancer, prostate cancer, endometrial cancer, bladder cancer,kidney cancer, esophageal cancer, squamous cell cancer, uveal melanoma,follicular lymphoma, renal cancer, cervical cancer, gastric cancer,astrocytic cancer, lung cancer, ovarian cancer, and colorectal cancer.5. The method of claim 4, wherein the cancer is selected from the groupconsisting of breast cancer, prostate cancer, lung cancer, ovariancancer, and colorectal cancer.
 6. A method for treating cancer in acancer patient comprising administering an effective amount of anantibody or antibody fragment to the patient, wherein (a) the antibodycomprises a heavy chain and a light chain, wherein the heavy chaincomprises the amino acid sequence of SEQ ID NO:11 and the light chaincomprises the amino acid sequence of SEQ ID NO:44; and (b) the cancer isselected from the group consisting of breast cancer, prostate cancer,endometrial cancer, bladder cancer, kidney cancer, esophageal cancer,squamous cell cancer, uveal melanoma, follicular lymphoma, renal cancer,cervical cancer, gastric cancer, astrocytic cancer, lung cancer, ovariancancer, and colorectal cancer.
 7. The method of claim 6, wherein thecancer is selected from the group consisting of breast cancer, prostatecancer, lung cancer, ovarian cancer, and colorectal cancer.
 8. A methodfor treating cancer in a cancer patient comprising administering aneffective amount of an antibody or antibody fragment to the patient,wherein (a) the antibody or antibody fragment is an antibody or antibodyfragment that competes with a reference antibody for binding to theextracellular domain of human c-fms of SEQ ID NO:1, and wherein thereference antibody comprises: (i) a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:77; and (ii) a lightchain variable domain comprising the amino acid sequence of SEQ IDNO:110; and (b) the cancer is selected from the group consisting ofbreast cancer, prostate cancer, endometrial cancer, bladder cancer,kidney cancer, esophageal cancer, squamous cell cancer, uveal melanoma,follicular lymphoma, renal cancer, cervical cancer, gastric cancer,astrocytic cancer, lung cancer, ovarian cancer, and colorectal cancer.9. The method of claim 8, wherein the cancer is selected from the groupconsisting of breast cancer, prostate cancer, lung cancer, ovariancancer, and colorectal cancer.
 10. The method of claim 1, wherein thecancer is breast cancer, prostate cancer or lung cancer.
 11. The methodof claim 4, wherein the cancer is breast cancer, prostate cancer or lungcancer.
 12. The method of claim 6, wherein the cancer is breast cancer,prostate cancer or lung cancer.
 13. The method of claim 8, wherein thecancer is breast cancer, prostate cancer or lung cancer.